New unsymmetrical bolaamphiphiles: Synthesis, assembly with DNA, and application for gene delivery

Article abstract of DOI:10.1021/bc100334t

The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these vectors is the poor structural control of their DNA complexes. In the present work, following new design we synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure. The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ～100 nm at low N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled small size and high transfection efficiency.

Full text of DOI:10.1021/bc100334t

2110
Bioconjugate Chem. 2010, 21, 2110–2118
New Unsymmetrical Bolaamphiphiles: Synthesis, Assembly with DNA, and
Application for Gene Delivery
Namrata Jain, Youri Arntz, Vale´rie Goldschmidt, Guy Duportail, Yves Me´ly, and Andrey S. Klymchenko*
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Universite´ de Strasbourg, Faculte´ de Pharmacie,
74, Route du Rhin, 67401 ILLKIRCH Cedex, France. Received July 23, 2010; Revised Manuscript Received September 20, 2010
The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on
lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these
vectors is the poor structural control of their DNA complexes. In the present work, following new design we
synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine
head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup
is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around
DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis
data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure.
The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ∼100 nm at low
N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to
neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or
spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when
either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization
is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results
show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that
further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled
small size and high transfection efficiency.
vectors based on lipids are highly attractive, since they can
deliver large quantities of genetic information and are weakly
immunogenic (8). A variety of cationic lipids for gene delivery
has been developed in recent decades (8-19). Their complexes
with DNA (lipoplexes) show high transfection efficiency in vitro,
though their application for gene therapy in vivo remains a
challenge. One of the key problems of cationic lipids is the poor
structural control of their lipoplexes (10, 20). Indeed, symmetric
lipid bilayers made of cationic lipids interact with DNA by
forming infinite sandwich-like structures of high polydispersity
(Figure 1) (10).
In this respect, unsymmetrical bolas bearing positively
charged and neutral head groups could be an attractive alterna-
tive to the cationic lipids. These molecules could generate
asymmetric membranes (in form of vesicles or nanotubes)
having positively charged inner and neutral outer surfaces. In
such a case, the inner membrane surface can be used to wrap
the DNA molecule (Figure 1), while the outer neutral membrane
surface, being inert to DNA, would prevent further oligomer-
ization of the complex and could be utilized for efficient
exposure of the biological signal for specific targeting. The
examples of successful application of bolas for gene delivery,
which has been shown only in the recent years, include synthetic
unsymmetrical bolas (21-25) and archaebacteria-derived
lipids (26, 27). Among unsymmetrical bolas, the most promising
were those bearing a sugar (gluconic or lactonic acid) residue
on one side and mono- or oligo-cationic ammonium-based group
on the other (21, 23). Moreover, the galactose-bearing bolas
showed some specificity to HepG2 cell line, expressing a
galactose receptor of endocytosis (21, 22).
INTRODUCTION
The vast majority of natural and synthetic lipids studied so
far are “monopolar” amphiphiles, that is, molecules presenting
one polar headgroup and generally two hydrophobic chains.
However, bipolar amphiphiles, which are analogues of lipids
presenting polar groups at the two opposite sides of the
hydrophobic chain(s), so-called bolaamphiphiles (bolas), became
the matter of intensive research only in the recent years (1). A
remarkable feature of bolas is that in contrast to bilayer
membranes formed by lipids they can form monolayer mem-
branes (Figure 1), having bolas with membrane-spanning
hydrophobic chains, which are believed to be responsible for
enhanced physical stability of bola membranes (1, 2). In nature,
they are mainly present in archaebacteria and ensure the integrity
of the bacterial membrane at high temperature (90 °C) and low
pH (1-1.5) (3, 4). Membranes made from synthetic bolas,
forming either nanotubes or vesicles, can also show an
exceptional stability, some of them being stable even in the dry
phase (5, 6) or in some organic solvents (7). Unsymmetrical
bolas, bearing two different polar head groups, assembled in a
parallel fashion can form asymmetric membranes, so that the
two opposite sides of the monolayer membrane present different
functional groups (Figure 1) (5, 6). One of the attractive
applications of bolas, which is still in its infancy stage, is gene
delivery, a domain for which “monopolar” cationic lipids and
other cationic agents are currently maintaining a dominant
position.
Gene delivery, which is a main concern in gene therapy, relies
heavily on development of new delivery vectors. Nonviral
In the present work, three new bolaamphiphiles were syn-
thesized, bearing neutral sugar and dicationic ornithine head
groups, connected by different hydrophobic spacers. We showed
* To whom correspondence should be addressed. E-mail:
andrey.klymchenko@unistra.fr. Fax: +33 368 854313. Tel: +33 368
854255.
10.1021/bc100334t  2010 American Chemical Society
Published on Web 10/14/2010

Unsymmetrical Bolaamphiphiles for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 11, 2010 2111
Figure 1. Lipids and bolaamphiphiles assembly with DNA. (A) Schematic presentation of bilayers formed from cationic lipids and further formation
of DNA complexes, so-called lipoplexes. (B) Assembly of bolaamphiphiles into asymmetric membranes and formation of DNA complexes, so-
called bolaplexes.
that the nature of spacer defines the bola self-assembly, the
interaction with DNA and the morphology of the obtained
complexes. Moreover, some bolas can transfect efficiently
COS-7 cells in the presence of either a helper lipid (DOPE) or
an endosomolytic reagent (chloroquine) (28). The latter indicates
that the key barrier for gene delivery with the present bolas
resides in the endosomal escape. This work presents new
bolaamphiphile-based nonviral vectors and provides insights for
further improvements of their efficiency.
(m, 4H), 1.47-1.38 (m, 18H), 1.37-1.16 (m, 22H). LC-MS
+
(m/z): found [M + 1]+ ) 491.2; calcd for C38H65N3O8
C10H18O4 (2Boc) ) 491.2.
-
(1-{4-[15-(2-Amino-ethylcarbamoyl)-pentadecyloxy]-phenyl-
carbamoyl}-4-tert-butoxycabonylamino-butyl)-carbamic Acid
tert-Butyl Ester (4). Methyl ester of 3 (0.7 mmol, 0.5 g) was
substituted with ethylenediamine (150 mmol, 10 mL) for 72 h
at 70 °C. Then the excess of ethylenediamine was evaporated,
50 mL of water was added and the product 4 was filtered (0.45
g, 87%). 1H NMR (CDCl3, 300 MHz): δ 8.45 (s, 1H), 7.42 (d,
2H), 6.82 (d, 2H), 6.05 (s, 1H), 5.30 (s, 1H), 4.75 (s, 1H), 4.37
(s, 1H), 3.91 (t, 2H), 3.42-3.25 (m, 3H), 3.16-3.02 (m, 1H),
2.84 (t, 2H), 2.17 (t, 2H), 2.06-1.92 (m, 4H), 1.79-1.7 (m,
2H), 1.67-1.54 (m, 4H), 1.48-1.37 (m, 18H), 1.37-1.20 (m,
20H). LC-MS (m/z): found [M + 1]+ ) 720.4; calcd for
MATERIALS AND METHODS
All chemicals and solvents for synthesis were purchased from
Sigma-Aldrich. Mass spectra were measured using Mass
Spectrometer Mariner System 5155. LC-MASS was performed
1
on Agilent, 1956 B/MSD. H NMR spectra were recorded on
+
Bruker 300 MHz spectrometer.
C39H69N5O7 ) 720.4.
N-Boc-4-aminophenol (1). 4-Aminophenol (4 g, 36.7
mmol) and Boc2O (8 g, 36.7 mmol) were dissolved in 30
mL of THF and left stirring overnight. Then 100 mL of water
was added and the product was extracted with ethyl acetate
(3 × 50 mL) and further dried with sodium sulfate. After
solvent evaporation in vacuo, the crude product 1 was used
without further purification.
[4-tert-Butoxylamino-4-(4-{15-[2-(2,3,4,5,6-pentahydroxy-
hexanoylamino)-ethylcabamoyl]-pentadecyloxy}-phenylcar-
bamoyl)-butyl]-carbamic Acid tert-Butyl Ester (5). D-gluconic
acid δ-lactone (0.23 mmol, 0.04 g) was added to solution of 4
(0.11 mmol, 0.1 g) in 15 mL of methanol. Then, DIEA (1.4
mmol, 0.24 mL) was added and the reaction mixture was stirred
at 70 °C for about 24 h. Solvents were evaporated in vacuo.
Compound 5 (0.09 g, 90%) was crystallized from methanol.
1H NMR (CD3OD, 300 MHz): δ 7.42 (d, 2H), 6.87 (d, 2H),
4.21 (s, 1H), 4.18-4.06 (m, 2H), 3.96 (t, 2H), 3.83-3.6 (m,
5H), 3.09 (t, 2H), 2.19 (t, 2H), 1.86-1.54 (m, 10H), 1.48-1.41
(m, 18H), 1.39-1.26 (m, 22H). LC-MS (m/z): found [M + 1]+
16-(4-tert-Butoxycarbonylaminophenoxy)-hexadecanoic Acid
Methyl Ester (2). Boc-protected 4-aminophenol (1) (1.8 g, 8.6
mmol) and 16-bromohexadecanoic acid methyl ester (3 g, 8.6
mmol, prepared by methylation of the acid in methanol and
thionyl chloride) were dissolved in 10 mL of DMF and
supplemented with 1.8 g (13 mmol) of potassium carbonate.
After 12 h of stirring at room temperature (RT), the solution
was poured in water (50 mL) and the product was extracted
with dichloromethane (3 × 30 mL) and dried with sodium
sulfate. Then the solvent was removed and the product 2 (1.6
+
) 898.57; calcd for C45H79N5O13 ) 898.57.
16-[4-(2,5-Diamino-pentanoylamino)-phenoxy]-hexadecanoica-
cid[2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-ethyl]-amide (Orn-
C16-G). Boc was removed from 50 mg of 5 using 0.5 mL TFA
and 3% H2O (1 h) to get the final product Orn-C16-G (27 mg,
1
g, 40%) was crystallized from ethanol. H NMR (CDCl3, 300
90%). LC-MS (Bruker, HTCultra) (m/z): found [M + 1]+
)
MHz): δ 7.24 (d, 2H), 6.82 (d, 2H), 6.4 (s, 1H), 3.91 (t, 2H),
+
698.4; calcd for C35H64N5O9 ) 698.4.
3.67 (s, 3H), 2.31 (t, 2H), 1.82-1.58 (m, 4H), 1.55-1.48 (m,
9H), 1.46-1.2 (m, 24H). LC-MS (m/z): found [M + 1]+
378.2; calcd for C28H47NO5+-C5H9O2 (Boc) ) 378.3.
)
Fluorescence Measurements. Absorption spectra were re-
corded on a Cary 400 spectrophotometer (Varian) and fluores-
cence spectra either on FluoroMax 3.0 or Fluorolog (Jobin Yvon,
Horiba) spectrofluorimeters. All the emission spectra were
corrected from the background signal of the corresponding blank
(corresponding solution without the fluorescent dye). For
measurements with 1,8-ANS, 50 nM of the dye dissolved in
20 mM MES buffer (pH 7.4 or 6.0) solution was titrated with
increasing quantities of the corresponding bola, added from stock
solutions. All spectra were recorded 2 min after each addition.
In the ethidium bromide (EtBr) exclusion assay, an aliquot of
CT-DNA (final concentration 20 µM) was added to the solution
of EtBr (0.4 µM). After 2 min, increasing quantities of the
corresponding bola were added from stock solutions (in DMF
with 10% of water). Fluorescence intensity recorded at 600 nm
(excitation at 550 nm) was recorded 2 min after each addition
of bola aliquot.
16-[4-(2,5-Bis-tert-butoxycaronylamino-pentanoylamino)-
phenoxy]-hexadecanoic Acid Methyl Ester (3). Boc was re-
moved from 2 (3.14 mmol, 1.5 g) by treating with 5 mL TFA
and 3% H2O for 1 h at RT. The solvent was evaporated and
1.54 g (3.1 mmol) of the obtained deprotected amine was
coupled with 2,5-bis-tert-butoxycarbonyl amino-pentanoic acid
(3.57 mmol, 1.15 g) using BOP (3.44 mmol, 1.52 g), HOBt
(4.3 mmol, 0.58 g), and DIEA (12.6 mmol, 2.2 mL) in DMF
(15 mL) and dichloromethane (30 mL). The mixture was stirred
overnight at RT. Then, solvent was evaporated and product was
crystallized from acetonitrile. The precipitate was filtered off
and dried to give compound 3 (1.6 g, 76%). 1H NMR (CDCl3,
300 MHz): δ 8.41 (s, 1H), 7.41 (d, 2H), 6.81 (d, 2H), 5.25 (s,
1H), 4.73 (s, 1H), 4.39 (s, 1H), 3.9 (t, 2H), 3.65 (s, 3H), 3.4 (s,
1H), 3.09 (s, 1H), 2.28 (t, 2H), 1.83-1.69 (m, 4H), 1.67-1.53

2112 Bioconjugate Chem., Vol. 21, No. 11, 2010
Jain et al.
Figure 2. Chemical structures of bolaamphiphiles.
Dynamic Light Scattering (DLS) and ꢀ-Potential Mea-
surements. The bolaplexes were prepared in 20 mM MES at
pH 7.4 by mixing equal volumes of the solutions of calf thymus
DNA (CT-DNA) or pCMV-Luc plasmid (pDNA) and the
corresponding bolas. The bola solutions were prepared by adding
aliquots of their stock solutions (in DMF with 10% of water)
to the buffer. The final DNA concentration (expressed in
phosphate) was 20 µM, while the bolas concentration was
adjusted according to a desired N/P ratio. The N/P ratio between
bolas and pDNA was expressed as the molar ratio between all
the protonable amino groups of the bolas and the phosphate
groups of the DNA. The DLS measurements were performed
after 30 min of incubation at room temperature. The average
size of the complexes was determined with a Zetasizer Nano
ZS (Malvern Instruments) with the following specifications:
sampling time, 30 s; medium viscosity, 0.8872 cP; refractive
index (RI) medium, 1.33; RI particle, 1.590; scattering angle,
90°; temperature, 25 °C. For particle analysis, the statistics based
on particle volume and particle number were presented. For
ꢀ-potential measurements, the same sample preparation was
done using CT-DNA at 30 µM concentration. The instrumental
specifications were the same, accounting for a solvent dielectric
constant of 78.5.
Gel Electrophoresis. The bolaplexes were prepared at
different N/P ratios using 60 µM pCMV-Luc plasmid (final
phosphate concentration), followed by 30 min incubation at
room temperature. Four microliters of loading dye (Lonza, 6×)
was added to 20 µL of the prepared complexes and then 12 µL
of this mixture was loaded on 0.9% agarose gel prepared with
0.5X TAE (tris-acetate-ethylenediaminetetraacetic acid) buffer.
In parallel, a 10 kbp DNA ladder (Lonza) was loaded on the
gel. Electrophoresis was carried out with 0.5X TAE buffer at a
constant voltage of 100 mV. DNA bands were visualized by
UV trans-illuminator (GeneGenius, Syngene) after coloration
with EtBr (0.5 µg/mL) for 15 min.
with 10% fetal bovine serum (FBS, Lonza) and 100 U/ml of
penicillin and streptomycin (Gibco-Intvitogen) at 37 °C in a
humidified atmosphere containing 5% CO2. Cells were seeded
at a density of 1 × 105 cells in a 24-well plate, 24 h before
transfection.
Transfection was done by using bola/pDNA (pCMV-Luc
plasmid, 1 µg/well) complexes at different N/P ratios in serum-
free Opti-MEM (Gibco-Invitrogen) or complete culture medium
(DMEM containing 10% of FBS). For this purpose, bolaplexes
were prepared in MES buffer (pH 7.4) as for DLS measurements
and then added to the cells in Opti-MEM. Chloroquine (100
µM) or DOPE (1:1 molar ratio with respect to bola) was used
to enhance transfection efficiency in some formulations. In
formulations with DOPE, a mixture of DOPE and a correspond-
ing bola in ethanol was evaporated in a round-bottom flask to
obtain a film. Then, MES buffer (pH 7.4) was added and the
samples were hydrated overnight at RT. Then, the samples were
vortexed vigorously for 1 min and further sonicated in an
ultrasonic bath for 15 min. The obtained suspensions were mixed
with an equal volume of pDNA in the buffer to obtain a desired
N/P ratio. All formulations were incubated at RT for 30 min
before addition to the cells. After 3 h of incubation of cells
with bolaplexes at 37 °C, 10% of FBS was added to serum-
free samples, while for all the samples with chloroquine the
transfection medium was replaced with fresh complete culture
medium and then cells were cultured for another 45 h. Total
incubation time for all samples was 48 h. Luciferase gene
expression of lysed cells was quantified using a commercial kit
(Invitrogen) and a luminometer (CentroXS3 LB 960, BER-
THOLD Technologies). Results were expressed as relative light
units integrated over 10 s per mg of total cell protein lysate
(RLU/mg of total protein). The experiments were repeated six
times for serum-free samples and three times for samples with
serum. The total protein was determined by BC assay (Protein
Quantitation Kit, Interchim) using Cary 4000 spectrophotometer.
As a positive control in the transfection measurements, com-
mercially available agent jetPEI (Polyplus) was used following
protocols provided in the kit.
AFM Measurements. AFM measurements were performed
using the Solver Pro-M (NT-MDT) instrument. The measure-
ments were performed in liquid phase (MES buffer pH 7.4) by
using the tapping mode. The cantilevers used were NSG03 type
(NT-MDT) with a typical spring constant of 1.7 N/m, a
resonance frequency of 32 kHz in liquid, and a tip curvature
radius of 10 nm. Images were acquired with a resolution of
512 × 512 and a scan rate of 2 Hz. The samples were prepared
as for DLS measurements, then 100 µL of the solution was
deposited on the freshly cleaved mica followed by the addition
of 10 µL of a MgCl2 stock solution to obtain a final concentra-
tion of 10 mM. The measurements were performed 10 min after
sample preparation.
Cytotoxicity of bolas was evaluated using 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-
Aldrich) assay (29). COS-7 cells were seeded in a 24-well plate
at 1 × 105 cells per well and after 24 h of incubation at 37 °C
supplemented with bolas at different concentrations or jetPEI
(150 µM, expressed as concentration of nitrogen residues) in
serum-free Opti-MEM. After incubation for 3 h at 37 °C, 10%
of FBS was added and cells were cultured for another 45 h.
Cells were washed with phosphate buffer saline (PBS) and
incubated with serum free medium containing 0.5 mg/mL MTT
for 3 h at 37 °C. Media was discarded and formazan crystals
were resuspended in 0.5 mL MTT solvent (Sigma-Aldrich).
Absorbance of formazan solution was measured at 570 nm with
Transfection and Cytotoxicity. COS-7 cells (African Green
Monkey kidney fibroblast cell line) were grown in Dulbecco’s
modified Eagle’s medium (Gibco-Intvitogen), supplemented

Unsymmetrical Bolaamphiphiles for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 11, 2010 2113
Scheme 1. Synthesis of Bola Orn-C16-G
respect to the background at 690 nm using Cary 4000 spectro-
photometer. Cell viability was expressed as relative absorbance
(%) of the sample versus control cells.
7.4 and 6.0 were tested, which could alter the protonation of
the amino groups of bolas in the self-assembled state. It can be
seen from Figure 3 that for Orn-C16-G at pH 7.4, fluorescence
intensity grows rapidly with bola concentration showing begin-
ning of saturation at higher concentrations. In contrast, at pH
6.0, a moderate increase in fluorescence intensity is followed
by rapid increase at concentrations >20 µM. These data suggest
that the critical aggregation concentration (CAC) at pH 7.4 is
too low to be detected with this method, while at pH 6.0 it is
clearly much larger being close to 20 µM. We could speculate
that the self-assembly could be accompanied by a partial
deprotonation of bola amino groups, so that lower pH may favor
disassembly (i.e., higher CAC). In contrast, for bola Orn-C20-G
rapid increase of 1,8-ANS fluorescence intensity was observed
both at pH 7.4 (see Figure S1 in Supporting Information) and
6.0 (Figure 3). This indicates that CAC of Orn-C20-G is much
RESULTS AND DISCUSSION
Design and Synthesis. The basic design of bolas relies on
one neutral group and one dicationic group connected by a
hydrophobic spacer. As neutral group we selected a sugar
residue, which is biocompatible and relatively inert. The
dicationic headgroup responsible for DNA binding was the
ornithine residue (Orn), featuring two amino groups separated
by a 4-carbon atom distance. This distance ensures protonation
of both amino groups at neutral pH and thereby efficient
interaction with negatively charged DNA. On the basis of this
design, three new bolaamphiphiles were synthesized, presenting
different hydrophobic spacers: (a) a dipeptide composed of end-
substituted amino acids of 8- and 12-carbon atoms, Orn-C8-
C12-G; (b) a hydrophobic linker composed of an alkyl chain
of C16-carbon atoms and an aromatic unit, Orn-C16-G; and
(c) a long (20-carbon atoms) alkyl chain, Orn-C20-G (Figure
2). This variation of the hydrophobic spacer could tune the
hydrophobic, H-bonding and π-stacking interactions between
bolas. An example of synthesis of Orn-C16-G is presented in
Scheme 1. Initially, the amino group of 4-aminophenol was
protected with Boc-group (1) and its hydroxyl was further
reacted with methyl 16-bromohexadecanoate. Then, the amino
group in the obtained conjugate 2 was further deprotected and
substituted with Boc-protected ornithine. The obtained conjugate
3 was further reacted with ethylene diamine giving 4. The latter
was then substituted with D-gluconic acid δ-lactone followed
by Boc-removal, affording final bola Orn-C16-G. The yields
of the individual steps were systematically >50% and the
obtained final bolas in 100-200 mg quantities were sufficiently
pure according to NMR, LC-MS and HPLC techniques (see
also Supporting Information).
Self-Assembly. 1,8-ANS dye is poorly fluorescent in water,
while on binding to lipid membranes it becomes strongly
fluorescent (30). In the present work, we used this probe to study
the concentration-dependent bolas assembly. Solutions at pH
Figure 3. Fluorescence intensity of 1,8-ANS (50 nM) as a function of
bolas concentration. (A) Orn-C16-G in MES buffer at pH 7.4 and 6.
(B) Three different bolas at pH 6.

2114 Bioconjugate Chem., Vol. 21, No. 11, 2010
Jain et al.
Table 1. Dynamic Light Scattering Data of Bolas and Bolaplexes in Aqueous Buffer (pH 7.4)
volume analysis
sample
diameter, nm
amount, %
number analysis mean diameter, nm
73
Orn-C16-G
bola (100 µM)
110
1450
109
88
12
95
bola/pDNA N/P 1.2 (12 µM bola)
bola/pDNA
90
152
92
45
N/P 2 (12 µM bola)
510
1550
870
140
1050
97
55
95
100
15
85
bola/pDNA N/P 2 (20 µM bola)
bola/pDNA N/P 5 (50 µM bola)
bola (100 µM)
1400
925
110
bola/pDNA
20
90
N/P 1.2 (12 µM bola)
930
970
80
Orn-C20-G
bola/pDNA N/P 2 (12 µM bola)
bola/pDNA N/P 2 (20 µM bola)
bola/pDNA N/P 5 (50 µM bola)
bola/DOPE (100 µM bola)
100
100
100
30
70
100
20
900
1200
1069
140
1370
1090
220
1120
220
Orn-C16-G/DOPE (1:1)
bola/DOPE/pDNA N/P 2 (20 µM bola)
bola/DOPE/pDNA N/P 3 (30 µM bola)
218
190
200
800
80
bola/DOPE/pDNA N/P 5 (50 µM bola)
1440
100
1350
lower than that of Orn-C16-G, probably because of its hydro-
phobic spacer, which is longer and does not contain bulky
aromatic group, thus ensuring tight bola packing. Finally, Orn-
C8-C12-G showed relatively weak increase in the fluorescence
of 1,8-ANS with increasing concentration suggesting that it does
not assemble at the studied concentration range. The latter result
is probably connected to the presence of an amide group within
the bola hydrophobic spacer, which may strongly diminish its
hydrophobic interactions and thus increase its CAC.
The information about molecular order of bolas assemblies
can be provided from fluorescence anisotropy of a rod-shaped
molecule TMA-DPH (31). The observed values of fluorescence
anisotropy for Orn-C16-G and Orn-C20-G assemblies at 100
µM are relatively high (0.30-0.32) at 20 °C and correspond to
a rigid and highly ordered environment similar to lipid
membranes in solid gel phase (0.32) for DPPC at 20 °C (data
not shown) (32). These high values of fluorescence anisotropy
suggest that bolas are probably organized in monolayers with a
transmembrane spanning orientation, thus excluding possible
bolas bilayer structure presenting a U-shaped conformation, as
reported for some other bolas (33).
by their ability to self-assemble. Finally, a mixture of Orn-C16-G
with a helper lipid DOPE (1:1 molar ratio with respect to bola),
which was important in our transfection experiments (see
below), showed complex formation at slightly higher N/P ratio,
being completed at N/P 3 (see Figure S2 in Supporting Infor-
mation).
The DNA condensation within the bolaplexes can be followed
by the ethidium bromide (EtBr) exclusion assay (34). This dye
when intercalated into DNA is highly fluorescent, while after
DNA condensation it is expelled from its intercalation site, thus
resulting in a strong decrease of its fluorescence intensity (up
to 90%). For these studies, a linear DNA from calf thymus was
used as the model. According to our data, Orn-C8-C12-G
showed a relatively weak and rather linear decrease of EtBr
fluorescence intensity as a function of N/P ratio (Figure 5),
which is in line with the gel electrophoresis data showing its
According to DLS data, Orn-C16-G and Orn-C20-G at 100
µM concentration form a significant fraction of small structures
(around 100 nm, Table 1) in line with fluorescence data
suggesting self-assembly of bolas at these conditions. These
structures were stable for 1-2 h, while after longer incubation
some precipitation of the bolas was observed.
Interactions with DNA. To study interactions of bolas with
DNA, gel electrophoresis was first used. It can be seen from
Figure 4A that Orn-C8-C12-G forms complexes with firefly
luciferase plasmid DNA (pDNA) only at high N/P ratios (ratio
between cationic groups of bola and phosphate groups of DNA).
Indeed, only at N/P > 10 the pDNA band disappears. This
indicates a relatively weak affinity of this bola to DNA. In
contrast, Orn-C16-G (Figure 4B) and Orn-C20-G (Figure 4C)
form complexes with pDNA at N/P values close to 1.0-1.2. It
can be noted that Orn-C20-G shows a slightly stronger interac-
tion since the DNA band disappears at lower N/P ratio (1.2) as
compared to Orn-C16-G (1.6). By taking into account that all
bola molecules are bearing the same dicationic ornithine residue,
the stronger interaction of Orn-C16-G and Orn-C20-G with
DNA is probably connected with a higher hydrophobicity of
their spacers, which in turn favors their self-assembly and their
cooperative interaction with DNA. Thus, we can assert that the
interaction of the present bolas with DNA is controlled in part
Figure 4. Agarose gel electrophoresis (0.9%) of Orn-C8-C12-G (A),
Orn-C16-G (B), and Orn-C20-G (C) complexed with pDNA at different
N/P ratios. Bands at the left of gels (A) and (B) correspond to 10 kbp
DNA ladder.

Unsymmetrical Bolaamphiphiles for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 11, 2010 2115
Figure 5. Exclusion of EtBr from CT-DNA complexes with bolas at
different N/P ratios. The fluorescence intensity was normalized to 100%
of the initial intensity.
Figure 6. ꢀ-potential of nanostructures formed by bolas alone and by
their formulations with DOPE and CT-DNA in MES buffer (pH 7.4)
at different N/P ratios.
weak interaction with DNA. In contrast, both Orn-C16-G and
Orn-C20-G showed a sharp decrease of EtBr fluorescence at
N/P around 1. Thus, in accordance with the gel electrophoresis
data it is confirmed that these bolas bind strongly to DNA by
showing a significant level of DNA condensation. It can be also
noticed that, similarly to the gel electrophoresis data, Orn-C20-G
shows an EtBr fluorescence decrease at lower N/P ratio as
compared to Orn-C16-G. Moreover, the fluorescence decrease
is stronger for the former bola (ca. 85% for Orn-C20-G as
compared to ca. 70% for Orn-C16-G). Thus, we can conclude
that Orn-C20-G presents a stronger affinity to DNA and
condense it more significantly as compared to Orn-C16-G. This
conclusion corroborates with the observed stronger ability of
Orn-C20-G to self-assemble without DNA evidenced by 1,8-
ANS data. Since Orn-C8-C12-G shows a poor affinity to DNA
and almost a negligible DNA condensation ability, we excluded
this compound from further characterization, focusing only on
Orn-C16-G and Orn-C20-G bolas.
Structural Characterization of Bolaplexes. To estimate the
size of the obtained bolaplexes, DLS technique was used.
Initially, the bolaplexes at different N/P ratios were prepared
by direct addition of increasing quantities of Orn-C16-G from
stock to pDNA solution. It was observed that at low N/P ratios,
the particle size was relatively small (around 100 nm), while
above N/P 1.2, their size increased rapidly reaching a maximum
at N/P 2 (700 nm) and then further decreases until N/P 5 (see
Supporting Information). In a second approach, the bolaplexes
were prepared by mixing equal volumes of bolas and DNA
aqueous solutions, followed by 30 min incubation at RT. This
method gave similar DLS results for low N/P ratios (1.2 and
2): relatively small particles at N/P ) 1.2 and much larger at
N/P ) 2 (Table 1). It is important to note that for Orn-C16-G
at lower concentration the bolaplexes at N/P 2 (30 min
incubation) showed much smaller sizes (150 nm). In contrast,
after 5 min of incubation, Orn-C16-G/pDNA complexes pre-
sented similar small sizes (ca. 150 nm) for both low and high
bola concentrations. Thus, the higher bola concentration prob-
ably favors aggregation of bolaplexes into larger particles. At
N/P 5, this preparation method (with 30 min incubation) also
gave relatively large particles as for N/P 2. The observed
discrepancy between the two methods of preparation for N/P 5
is probably related to the incubation time, since in the first
approach the measurements were done 5 min after each addition.
The DLS data obtained with Orn-C20-G were similar to those
with Orn-C16-G (Table 1), so that bolaplexes were small at
N/P ratio 1.2, while their size increased significantly at higher
N/P ratios. Moreover, for Orn-C16-G/DOPE mixture (1:1), a
similar increase in the size of the bolaplexes was observed at
higher N/P ratios (Table 1). The observation of larger complexes
at higher N/Ps differs clearly from behavior of polyplexes and
lipoplexes. Indeed, the latter at N/Ps close to 1 show relatively
large complexes because of neutral charge of the complex, while
at higher N/Ps they are much smaller because of their strong
positive charge (35-37). Interestingly, our observations are in
line with recent reports on other bolas featuring cationic group
on one end and a neutral sugar residue on the other (23, 24).
Therefore, we can suggest that the increase in size of bolaplexes
at higher N/Ps could be a special feature of this type of bolas,
which is probably because their bolaplexes do not become
positively charged at higher N/Ps. This hypothesis was verified
by measuring the zeta potential of bolaplexes (Figure 6). The
bolas alone showed strongly positive ꢀ-potentials, while the
bolaplexes at low N/P ratios were negatively charged and
reached neutrality at N/P ratios around 3. At N/P 5, the observed
ꢀ-potential was positive, however, we suspect that it could be
due to excess of bola molecules that do not complex with DNA,
thus contributing to apparent positive potential values. For Orn-
C16-G/DOPE mixture (1:1), the ꢀ-potentials were systematically
more negative, though a clear neutralization of the complexes
was observed at N/P 5 (Figure 6). We expect that, as shown in
Figure 1B, the positively charged part of bola interacts with
DNA, while the neutral sugar residue is exposed at the bolaplex
surface. This structure may favor neutrality of the bolaplex at
higher N/Ps. Presence of DOPE does not change this tendency,
but only shifts the complex neutralization to higher N/P ratios.
To access the nanoscopic architecture of bolaplexes, we
performed atomic force microscopy (AFM) measurements in
liquid (buffer) phase on a mica surface. An efficient absorption
of the bolaplexes (N/P 2 or 1.2) at the mica surface was achieved
only in the presence of magnesium cations. This indicates that
the bolaplexes at the studied N/P ratios are negatively charged,
so that Mg2+ ions act as a link between bolaplexes and the
negatively charged mica surface. Orn-C16-G bolaplexes with
pDNA formed relatively small and spherical structures (Figure
7A). The average height and width of the particles were 12 and
120 nm, respectively, which correspond to an average particle
volume of about 1.7 × 105 nm3 and an equivalent spherical
particle diameter of 70 nm. This size is smaller than the one
observed by DLS for N/P 2 (12 µM bola). Several reasons may
explain this discrepancy. At first, the smaller size particles
observed by AFM is based on a particle by particle data analysis,
while DLS measures an average size with higher sensitivity to
larger particles. Indeed, the DLS data based on particle number

2116 Bioconjugate Chem., Vol. 21, No. 11, 2010
Jain et al.
Figure 7. AFM images of Orn-C16-G (A and B, N/P 2) and Orn-C20-G (C and D, N/P 1.2) bolaplexes with pDNA (A and C) and CT-DNA (B
and D). Images were obtained by tapping mode.
Figure 8. Transfection efficiency of the Orn-C16-G bolaplexes at different N/P ratios in COS-7 cells without (A) and with serum (B). Cells were
incubated in serum-free Opti-MEM (A) or complete culture medium, DMEM containing 10% of FBS (B) with a bolaplex composed of plasmid
DNA (1 µg per well), Orn-C16-G, and DOPE (when indicated). For some samples, the medium contained 100 µM chloroquine. After 3 h, 10% of
FBS was added to the serum-free samples, while for all the samples with chloroquine the transfection medium was replaced with fresh complete
culture medium. The luciferase activity quantification was performed after 48 h of incubation. Transfection efficiency determined from the luciferase
assay was expressed as RLU/mg of protein. The negative controls were nontreated cells (-pDNA) and those transfected with naked pDNA (pDNA).
The experiments were repeated six times for serum-free samples (A) and three times for samples with serum (B).
statistics is much closer to AFM data than those based on
volume analysis (Table 1). Moreover, DLS data provides a
hydrodynamic size of the particles, which is larger than the real
size. Finally, in AFM measurements, the particles are stabilized
by adsorption at the surface, while in DLS they are free in
solution and can further aggregate to form larger particles.
Bolaplexes of Orn-C16-G with CT-DNA showed high poly-
dispersity and formed both spherical and short rodlike structures
(Figure 7B). The presence of short rods of 8-10 nm height,
40-60 nm width, and 300-600 nm length could be explained
by the linear structure of CT-DNA that favored the formation
of elongated structures. In contrast, Orn-C20-G/DNA bolaplexes
at N/P 2 were very large and polydisperse, in line with DLS
data, which prompted us to study the bolaplexes at N/P 1.2.
Remarkably, the complexes at N/P 1.2 showed predominantly
rodlike structures for both pDNA and CT-DNA (Figure 7C and
D). However, in the case of pDNA, the observed rods were
relatively short, having height and width around 10 and 120
nm, respectively, while in the case of CT-DNA, they correspond
to very long fibers of 16 nm height and 80 nm width. The
formation of these “spaghetti-like” structures was previously
reported for cationic lipids (38). It was proposed that in this
kind of structure, the DNA molecule is located in the core, being
surrounded by the lipid membrane. Probably, Orn-C20-G/CT-
DNA bolaplexes present a similar structure, where single or
multiple DNA strands are surrounded by a monolayer of bola
molecules, similarly to the idealistic model in Figure 1B.

Unsymmetrical Bolaamphiphiles for Gene Delivery
Bioconjugate Chem., Vol. 21, No. 11, 2010 2117
Transfection Efficiency and Cytotoxicity. The transfection
efficiency of bolaplexes was in serum-free and serum-containing
media for the different formulations by using the luciferase
expression assay (Figure 8). For serum-free samples (Figure 8A),
considerable transfection efficiency was observed for Orn-C16-G
bolaplexes at N/P 2, 3, and 5 only in the presence of helper lipid
DOPE or the endosomolytic reagent chloroquine (28). The better
efficiency observed in the presence of DOPE is in line with
previously reported data concerning other bolaamphiphiles (21, 23).
DOPE probably improves the fusion of bolaplexes with cellular
and endosomal membranes (39, 40). However, the strong effect
of chloroquine on bolaplexes delivery is reported for the first time.
This result clearly shows that the key barrier for internalization of
bolaplexes is related to the endosomal escape. In the presence of
serum, the transfection efficiency decreased significantly for all
samples with DOPE (Figure 8B), which is in line with previous
reports showing that DOPE-containing lipoplexes are highly
sensitive to serum (41). In the presence of chloroquine, the effect
of serum was less important, and the transfection efficiency
increased systematically at higher N/P ratio. At N/P ) 5, the
sample with serum showed even slightly higher transfection
efficiency than that without serum (Figure 8). Similar results
were reported for lipoplexes, where higher N/Ps increased their
resistance to serum (42).
On the other hand, bolaplexes of Orn-C20-G displayed
relatively poor transfection efficiency for most of the formula-
tions, which can be related to their larger size and spaghetti-
like architecture (Figure 7). It should be noted that the observed
values of transfection efficiency for Orn-C16-G were signifi-
cantly lower than that for highly efficient commercial agent
jetPEI (3 × 108 RLU/mg protein in the present study), while
they were comparable with those recently reported for some
other bolaamphiphiles (21, 22). Taking into account that the
area of bolaamphiphiles as nonviral vectors is still poorly
explored, we believe that there is a lot of room for improvement
of their transfection efficiency. It will require optimization of
bola chemical structure to achieve better control on the size
and stability of the bolaplexes as well as on their ability to
escape from endosomes. This work is currently in progress and
will be reported in due course.
for a further development of new efficient gene delivery vectors
based on this original class of compounds.
ACKNOWLEDGMENT
This work was supported by Fondation pour la Recherche
Me´dicale (FRM). We thank the groups of J.-S. Remy and B.
Frisch for help with luminometry and DLS measurements. We
also thank G. Zuber for fruitful discussions.
Supporting Information Available: Procedures for synthesis
of Orn-C20-G and Orn-C8-C12-G. Some DLS data. Total
protein and MTT-based cytotoxicity assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
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