The effect of charge-reversal amphiphile spacer composition on DNA and siRNA delivery

Article abstract of DOI:10.1021/bc9005464

A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic chains are described for delivery of DNA and siRNA. Among them, the amphiphiles possessing a glycine spacer (e.g., B-GlyGly) showed effective DNA

Full text of DOI:10.1021/bc9005464

988
Bioconjugate Chem. 2010, 21, 988–993
The Effect of Charge-Reversal Amphiphile Spacer Composition on DNA
and siRNA Delivery
Xiao-Xiang Zhang,† Carla A. H. Prata,† Thomas J. McIntosh,‡ Philippe Barthe´le´my,§ and Mark W. Grinstaff*,†
Departments of Biomedical Engineering and Chemistry, Boston University, Boston, Massachusetts 02215, Department of Cell
Biology, Duke University Medical Center, Durham, North Carolina 27710, and Inserm, U-869, Universite´ de Bordeaux,
Bordeaux, F-33076, France. Received December 9, 2009; Revised Manuscript Received April 7, 2010
A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic
chains are described for delivery of DNA and siRNA. Among them, the amphiphiles possessing a glycine spacer
(e.g., B-GlyGly) showed effective DNA transfection in CHO and NIH 3T3 cells, as well as siRNA gene knockdown
in HepG2 and UASMC cells. Ethidium bromide quenching assays revealed that DNA was released the fastest
from the lipoplex of B-GlyGly in the presence of esterase. Also, X-ray diffraction results indicated that the DNA
was located between the adjacent lipid bilayers in the lipoplex of B-GlyGly. These distinct features appear to be
required for high transfection activity.
INTRODUCTION
Gene therapy has received significant attention in the past
decade due to its advantages over traditional therapies. The two
most common methods for gene delivery use either synthetic
(1-5) or viral (6-10) vectors. Compared to viral vectors,
nonviral vectors have lower toxicity and high load capacity and
are easy to synthesize. Consequently, there has been a significant
effort to develop and evaluate nonviral vectors, which include
cationic amphiphiles and polymers (11-15) and, more recently,
Figure 1. Amphiphiles previously studied.
anionic amphiphiles (16). Yet, synthetic vectors also have a
number of limitations including low transfection efficiencies
both in vitro and in vivo, significant cytotoxicity, and inactiva-
tion in the presence of serum. To overcome these limitations,
investigators are modifying the amphiphile structure, and for
small cationic amphiphiles, this entails alternative cationic head
groups, linkers, and hydrophobic moieties. Examples of different
cationic head groups explored include choline (17, 18), spermine
(19), di- and tripeptides (20, 21), carbohydrates (22, 23), and
nucleosides (24-32). With regard to the hydrophobic moiety, long
chain ester and ether linked hydrocarbons have been extensively
studied. The fluorinated analogues of cationic lipids, like DOTMA,
have also been explored, and these amphiphiles exhibited higher
transfection activities in vitro (33) and in vivo compared to their
hydrocarbon analogues (34). In addition, researchers have used
combinatorial approaches to screen large libraries of amphiphiles
in order to identify key structural components of the amphiphile
responsible for high transfection activity (35, 36).
Although significant research has been conducted regarding
the structure-property relationships of a wide range of cationic
lipids, only a few studies can be found that systematically
investigate the effect of spacers within the headgroup, and
include cationic lipids derived from betaine glycine (37), cationic
glycolipids (38), and cholesterol-based cationic lipids (39). These
studies, along with others, have begun to provide design
parameters for optimizing the chemical structure for efficient
nucleic acid delivery with minimal cytotoxicity.
In previous research in our lab, we found that a charge-
reversal amphiphile (Figure 1A) which possesses benzyl esters
at the end of alkyl chains showed high transfection activity (40).
The total charge of this amphiphile was capable of switching
from +1 to -1 when the benzyl esters are hydrolyzed. This
charge-reversal effect was implemented to facilitate the release
of DNA from the amphiphiles. In a separate study, we also
found that spacing of the cationic charges within the headgroup
in a series of peptide-based amphiphiles can afford improved
transfection performance with amphiphiles KWK-C14, KGK-
C14, and KGG-C14 showing higher transfection compared to
KK-C14 (Figure 1B) (21). To take advantage of the charge-
reversal effect, as well as to further understand how spacer
length and composition affect transfection efficacy, here we
present a study on a series of charge-reversal amphiphiles with
different spacers separating the headgroup from the hydrophobic
chains.
All the amphiphiles studied (Figure 2) possess a quaternary
ammonium as a cationic headgroup (B) and the same alkyl
chains ending with benzyl esters, but have spacers of different
lengths: two, five, or seven atoms. The nature of the spacers is
also varied: (1) the glycine and alanine spacers are relatively
rigid, hydrophilic, and capable of hydrogen bonding (B-Gly,
B-GlyGly, B-AlaAla; (2) the ethylene oxide spacers are flexible
and hydrophilic (B-EO, B-(EO)2); and (3) the alkyl chain
spacers are flexible and hydrophobic (B-(CH2)4, B-(CH2)7).
* Correspondence should be addressed to M.W.G. E-mail: mgrin@
RESULTS AND DISCUSSION
bu.edu.
† Boston University.
‡ Duke University Medical Center.
§ Inserm, U-869.
The synthetic routes to the different amphiphiles can be found
in Scheme 1, and a detailed procedure can be found in the
Supporting Information. In general, the different spacers
10.1021/bc9005464  2010 American Chemical Society
Published on Web 04/30/2010

Charge-Reversal Amphiphile Spacer Composition
Bioconjugate Chem., Vol. 21, No. 5, 2010 989
Figure 2. Amphiphiles with different spacers.
Scheme 1. Synthesis of the Cationic Amphiphiles
Figure 3. EtBr displacement assay showing the fluorescence intensity.
Top: as a function of amphiphile/DNA charge ratio. Bottom: as a
function of time in presence of porcine liver esterase (300 units/mL).
was removed in the presence of a catalytic amount of Pd(II) on
carbon at RT under 50 psi H2 for 16 h to afford the intermediate,
in 84% yield. The deprotected 2-(2-(dimethylamino)acetami-
do)acetic acid was then coupled with 2 followed by reaction
with CH3I to afford the B-GlyGly amphiphile as a viscous oil
in 80% yield. The characterization data for B-GlyGly, as well
as all of the amphiphiles, can be found in the Supporting
Information.
To determine whether the amphiphiles would bind DNA, a
standard ethidium bromide (EtBr)-DNA fluorescence quenching
assay was performed, as shown in Figure 3 (top). DNA binding
was observed for all the amphiphiles with similar affinity.
Among the amphiphiles, B-(CH2)4 and B-(CH2)7 showed the
strongest binding affinity with a ( charge ratio of 6:1, and B
bound the weakest with a charge ratio of 9:1. Next, the above
DNA/EtBr/amphiphile solution was incubated with esterase at
pH 7.4 (100 mM Tris, 100 mM NaCl buffer, 300 units/mL).
The intensity of fluorescence, which reflects the dissociation of
the amphiphile from the DNA as a consequence of the
hydrolysis of the terminal benzyl esters, increased at slightly
different rates for the amphiphiles, as shown in Figure 3
(bottom). DNA was released the fastest from the B-GlyGly
complex with around 30% fluorescence recovered in the first
30 min, while less than 10% fluorescence recovery was observed
for all the other complexes. During the 4 h period, fluorescence
intensity increased slowly and reached ∼20-35% for all other
complexes, except for B where the fluorescence did not change
significantly.
containing the headgroup were first synthesized and then coupled
to 1 or 2 to afford the desired products. As a representative
example, the synthesis of the B-GlyGly amphiphile is described
next. N,N-Dimethylglycine hydrochloride and glycine benzyl
ester hydrochloride were reacted in the presence of DCC and
DMAP at RT for 16 h. Benzyl 2-(2-(dimethylamino)acetami-
do)acetate was purified as yellow oil (76%). The benzyl group
Given their polar headgroup and long hydrophobic alkyl
chains, these amphiphiles are likely to form bilayer vesicles in
aqueous solution. Differential scanning calorimetry (DSC) traces
of hydrated amphiphiles showed phase-transition temperatures
between ∼-22 and -15 °C, except for B, which was 60.6 °C

990 Bioconjugate Chem., Vol. 21, No. 5, 2010
Zhang et al.
Figure 5. DNA transfection after 48 h in CHO (top) and NIH3T3
(bottom) cells as a function of amphiphiles and DNA molar ratio.
Lipofectamine 2000 was used as positive control. N ) 3, mean ( SD,
* p < 0.05.
Figure 4. Sizes of lipoplexes (nm) at pH 7.4 (top) and pH 5 (bottom).
N ) 3, mean ( SD.
Table 1. Phase Transition Temperatures of the Amphiphiles and
Repeat Periods without and with DNA
samples in the absence and presence of DNA. A hydrated vesicle
pellet was obtained for all samples, and oriented multilayers
were prepared for X-ray analysis by placing the pellets on a
curved glass substrate and incubating at 66% relative humidity.
All of the samples showed multiple reflection indexing as orders
of lamellar repeat periods, characteristic of lipid bilayers (Table
1).
The repeat periods of all samples in the absence of DNA
were between 5.4 and 7.6 nm, except for B-(CH2)7, which had
a period of 1.4 nm. This period was abnormally small and could
be a higher-order reflection from a larger repeat period. The
addition of DNA increased the repeat period for all of the
assemblies. A smaller increase between 0.1 and 0.6 nm was
observed for B, B-Gly, B-(CH2)4, B-(EO)2, and B-AlaAla,
while for B-GlyGly, we observed an increase in the period of
1.8 nm. This larger change would be consistent with a structural
model where a smectic phase was formed with the DNA chains
located between the adjacent lipid bilayers within the multila-
mellar liposome. This structural model has been reported for
complexes of DNA with DOTAP (45) and cationic triesters of
phosphatidylcholine (46). For the other samples, the repeat
periods did not change significantly, indicating that DNA was
not located within the lipid multilayers.
Transfection experiments using reporter gene, ꢀ-galactosidase
(ꢀ-gal, pVax-LacZ1, Invitrogen) were performed with CHO and
NIH 3T3 cells (Figure 5). Gene transfection results were
determined after 48 h as a function of cation/anion ratios (4:1,
8:1, 12:1, 16:1).
In CHO cells, amphiphiles B-Gly and B-GlyGly showed the
highest transfection activity, amphiphile B-AlaAla exhibited
moderate activity, and the other amphiphiles showed minimal
transfection efficiency. B-GlyGly was significantly more active
than the positive control, Lipofectamine 2000. However, both
amphiphiles B-Gly and B-GlyGly were less active than Lipo-
fectamine 2000 in NIH 3T3 cells, but still had higher transfection
efficiency compared to the other amphiphiles we tested. These
results showed that those amphiphiles containing spacers that
are hydrophilic, relatively rigid, and capable of hydrogen
bonding possessed the highest transfection activity. We also
repeating period (nm)
amphiphiles
Tm (°C)
without DNA
with DNA
B
B-Gly
B-EO
B-(CH2)4
B-GlyGly
B-(EO)2
B-(CH2)7
B-AlaAla
60.6
-15.4
-22.2
-17.3
-16.8
-19.6
-14.8
-19.3
5.9
5.4
5.7
7.6
6.1
6.4
1.4
6.4
6.2
5.6
6.7
7.8
7.9
6.5
5.8
7.0
(Table 1). We subsequently prepared liposome solutions of all
the amphiphiles by sonicating a hydrated film of the amphiphile
in an aqueous solution (see Supporting Information for details).
Dynamic light scattering measurements revealed that the sizes
of liposomes in the presence of DNA varied from 154 to 353
nm in pH 7.4 solution (100 mM NaCl, 100 mM Tris buffer).
The stability of the liposomes over time was examined by
measuring their sizes at 0, 1, 2, and 4 h (Figure 4 top). The size
of liposomes formed from B-Gly, B-(EO)2, and B-(CH2)7
remained almost constant during the 4 h period, while those of
other amphiphiles increased moderately. Given that these
increasing rates are very small, all the liposomes appeared to
be stable.
Since endocytosis is one of the major cell uptake pathways
for internalization of nonviral vectors (41-44), liposomes were
incubated in a solution (100 mM NaCl, 100 mM phosphate
buffer) of pH 5, which resembles the pH of the endosome, and
analyzed at 0, 1, 2, 4 h (Figure 4 bottom). Generally, the size
of liposomes at pH 5 is slightly smaller than those at pH 7.4.
During the 4 h period, all the liposomes were stable except for
the liposome formed with B-EO, which quickly increased in
size. The size increased from 264 to 468 nm in 1 h, and further
to 655 and 1428 nm in 2 and 4 h. These findings demonstrated
that the liposomes formed from the amphiphiles were stable
under pH 7.4 and mildly acidic conditions, except those formed
from B-EO.
Next, to characterize the supramolecular structures, X-ray
diffraction experiments were performed at 22 °C on all of the

Charge-Reversal Amphiphile Spacer Composition
Bioconjugate Chem., Vol. 21, No. 5, 2010 991
Figure 6. Structures of B-Gly-C16 and B-GlyGly-C16 and DNA
transfection after 48 h in CHO cells as a function of amphiphiles and
DNA molar ratio. Lipofectamine 2000 was used as positive control. N
) 3, mean ( SD.
Figure 7. siRNA transfection after 48 h in HepG2 cells (top) and
UASMC cells (bottom) as a function of amphiphiles and siRNA molar
ratio, NeoFX was used as positive control. N ) 3, mean ( SD.
prepared the B-AlaAla amphiphile, since this amphiphile is
similar to the B-GlyGly amphiphile but the spacer is slightly
more hydrophobic and rigid. The B-AlaAla amphiphile showed
moderate transfection activity but less than that observed with
the B-GlyGly amphiphile. To confirm that the benzyl ester
linkages on the hydrophobic chains (and the resulting charge-
reversal effect) were a contributing factor to the gene delivery
efficiency of these amphiphiles, we synthesized two additional
control amphiphiles with the Gly or GlyGly spacers, possessing
saturated C16 chains instead of the benzyl ester terminated
hydrophobic chains. As shown in Figure 6, both amphiphiles
displayed minimum transfection activity in CHO cells. This
result demonstrated the importance of the charge-reversal effect
on the transfection. MTS assays performed with all the
amphiphiles showed no significant cytotoxicity in the two cell
lines (see Supporting Information).
Next, siRNA transfections were performed with the base
amphiphiles B and the two best performing amphiphiles, B-Gly
and B-GlyGly, using the KDalert GAPDH assay (Ambion) with
HepG2 and UASMC cells (Figure 7) at four different cation/
anion ratios (1:1, 5:1, 10:1, 15:1). NeoFX was used as the
positive control. After 48 hrs of transfection, in HepG2 cells,
B and B-Gly achieved 50% gene knockdown, while B-GlyGly
only achieved 20% knockdown. In UASMC cells, B and B-Gly
achieved 50% gene knockdown, while B-GlyGly reached 60%
knockdown. These results were comparable to those obtained
with NeoFX.
the peptide spacers was similar to all the other amphiphiles,
but DNA was released the fastest from B-GlyGly in the presence
of esterase. Also, X-ray diffraction results indicate that DNA
is located between the adjacent lipid bilayers in the complex
formed with B-GlyGly. It is clear that many factors can
influence transfection efficiency and that small perturbations in
structure can have a significant effect. In this study, we found
that the best performing amphiphile has a peptide linker
separating the headgroup from the chains, complexes with DNA
to afford a bilayer structure, and forms lipoplexes of 200 nm in
diameter. This study highlights the importance and sensitivity
of the spacer group in DNA/amphiphile assembly, liposome
stability, and gene transfection activity. Continued studies with
different amphiphile compositions will facilitate the development
of a set of design requirements for efficient DNA and RNA
delivery.
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
(EB005658 to M.W.G. and GM27278 to T.J.M.).
Supporting Information Available: Detailed synthetic pro-
cedures, preparation of liposomes, lipoplexes, and DNA and
siRNA transfection protocols, and statistical analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
CONCLUSION
In summary, a series of amphiphiles with different spacers
within the headgroup were evaluated for DNA and siRNA
delivery. Among them, the amphiphiles possessing the relatively
rigid and hydrophilic glycine or alaine spacers, capable of
hydrogen bonding, showed effective DNA transfection in vitro.
As for siRNA transfection, B, B-Gly and B-GlyGly showed
gene knockdown. The binding affinity for the amphiphiles with
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