Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery

Article abstract of DOI:10.1016/j.bbamem.2011.04.020

Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n = 2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n = 3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.

Full text of DOI:10.1016/j.bbamem.2011.04.020

Biochimica et Biophysica Acta 1808 (2011) 2156–2166
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamem
Synthesis and characterization of degradable multivalent cationic lipids with
disulfide-bond spacers for gene delivery
Rahau S. Shirazi a, Kai K. Ewert b,c,d, , Cecilia Leal b,c,d, Ramsey N. Majzoub b,c,d
,
⁎
Nathan F. Bouxsein b,c,d, Cyrus R. Safinya b,c,d,
⁎
a
Chemistry and Biochemistry Department, University of California, Santa Barbara, CA 93106, USA
Department of Materials, University of California, Santa Barbara, CA 93106, USA
Department of Physics, University of California, Santa Barbara, CA 93106, USA
Molecular, Cellular & Developmental Biology Department, University of California, Santa Barbara, CA 93106, USA
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being
explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems,
synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their
efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We
present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5)
containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be
cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering
demonstrates that the initially formed lamellar phase of CMVLn–DNA complexes completely disappears when
reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the
intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with
efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial
reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be
used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their
transfection efficiency.
Received 29 December 2010
Received in revised form 4 April 2011
Accepted 12 April 2011
Available online 24 May 2011
Keywords:
Gene delivery
Cationic lipid
Cationic liposome
Nonviral
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
(particularly in vivo) and have resulted in gene therapy's first successes
[7], but they suffer from a number of drawbacks. A primary concern is
Gene therapy has the potential to address a wide variety of diseases
at their root cause. These include cancer, hereditary disorders, and
many others [1–4]. However, despite massive efforts to the contrary,
including numerous clinical trials worldwide [5,6], widespread thera-
peutic success has remained elusive. A key challenge for gene therapy
is to develop efficient and safe gene delivery methods. Viral vectors,
such as engineered adenoviruses and retroviruses, are very efficient
vector safety: in clinical trials, viral vectors have led to serious adverse
effects, owing to immunogenicity and non-specific integration of their
genetic cargo into the host genome [8–11]. Other limitations are the
small size of viral genomes and the comparatively difficult vector
production. Synthetic nonviral vectors, on the other hand, lack
immunogenic protein components, do not impose a size limit on the
nucleic acid to be transferred, and are more easily prepared and
modified. Their main limitation is their lower transfection efficiency
(TE, a measure of the amount of successfully transferred and
transcribed DNA) and the onset of toxicity with increasing cationic
lipid/nucleic acid mole ratios, which need to be improved in order to
compete with viral vectors [12–15].
Cationic liposome (CL)–nucleic acid (NA) complexes are one of the
main classes of nonviral vectors [13,16–20]. They form spontaneously
when cationic liposomes (typically containing a neutral lipid (NL)
as well as a cationic lipid) are combined with NAs. A large number
of cationic lipids have been synthesized [21–23] to enhance the
efficiency of CL–DNA complexes and uncover structure–TE relation-
ships, albeit with limited success. Thus, further development of
cationic lipid-based vectors is required to match the TE of viral
Abbreviations: Boc, tert-butoxycarbonyl; CL, cationic liposome; DEAD, diethylazo-
dicarboxylate; DIEA, N,N-diisopropylethylamine; DLS, dynamic light scattering; DOB,
3,4-di(oleyloxy)benzoic acid; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine;
DOTAP, 2,3-dioleoyloxypropyltrimethylammonium chloride; DTT, dithiothreitol; EtBr,
ethidium bromide; GSH, glutathione; L/D, cationic lipid/DNA weight ratio; NA, nucleic
acid; NL, neutral lipid; RLU, relative light units; SAXS, small-angle X-ray scattering;
TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TE,
transfection efficiency; TFA, trifluoroacetic acid; TLC, thin-layer chromatography;
TL/D, total lipid/DNA weight ratio; Φi, mole fraction of lipid i in the lipid mixture; σM
,
membrane charge density
⁎
Corresponding authors.
E-mail addresses: ewert@mrl.ucsb.edu (K.K. Ewert), safinya@mrl.ucsb.edu
(C.R. Safinya).
0005-2736/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamem.2011.04.020

R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
2157
vectors. Among the chemical approaches to improve the TE of CL–
DNA complexes, the synthesis of cationic lipids with multivalent head
groups has been particularly promising [24–30].
Supplementary data, Fig. S1. For X-ray samples and ethidium bromide
displacement assay, highly polymerized calf thymus DNA (Sigma-
Aldrich) was used. Luciferase plasmid DNA (pGL3 Control Vector,
Promega) for microscopy, dynamic light scattering, cytotoxicity, and
transfection assays was propagated in E. coli and isolated using a
Qiagen Giga Kit. Other chemicals were purchased from Sigma-Aldrich
unless otherwise stated and were used as received.
The development of cationic lipids with low cytotoxicity is an
important objective, especially for applications such as gene silencing
which require higher cationic lipid/nucleic acid ratios [27]. The
toxicity of CL–NA complexes is known to be due to the cationic
lipid component of the complex [27,31]. Thus, we hypothesized that
triggered disintegration of an appropriately designed, degradable
multivalent cationic lipid component of the CL–DNA complex upon
entry into the cytoplasm should reduce cytotoxicity. At the same
time, such lipids should retain high TE levels comparable to those of
recently synthesized non-degradable multivalent lipids [24–30]
because the key steps of lipid-mediated DNA delivery prior to
cytoplasmic release—DNA compaction, complex uptake and endoso-
mal escape [32,33]—will not be compromised by the lipid degradation
process.
2.2. General methods
NMR spectroscopy was carried out on Bruker Avance instruments
(200 MHz and 500 MHz). Detection of the spots in thin-layer
chromatography was achieved by UV absorption and ninhydrin
reagent (300 mg in 95 mL of 2-propanol and 5 mL of acetic acid) or
phosphomolybdic acid in ethanol (10 wt%). Silica gel (Merck) with a
mesh size of 200–425 was used for flash chromatography [51].
In order to test this hypothesis and potentially decrease CL-
induced cytotoxicity of lipid vectors, we prepared a series of cationic
lipids designed to quickly degrade in the cytoplasm. As we describe,
degradable multivalent lipids exhibit reduced toxicity with increasing
cationic lipid/nucleic acid mole ratio while maintaining the high TE
levels of previously synthesized multivalent lipids [24–30], consistent
with our hypothesis.
Cells maintain a high redox potential gradient between the
intracellular (reducing) environment and extracellular (oxidizing)
space. The main intracellular reducing agent is the short peptide
glutathione (GSH), which is continually recycled [34]. The intracel-
lular concentration of GSH varies for different tissues but can reach up
to 5 mM (e.g. in the liver) [35,36]. The redox potential gradient can be
exploited to trigger, e.g., the cleavage of disulfide bonds in molecules
once they reach the cytoplasm. This mechanism is used by biological
toxins [37] as well as in drug delivery and diagnostic imaging [38–42].
A few studies have also applied the concept to gene delivery vectors
[43–46], including CL–DNA complexes [47–50].
We report the convenient and efficient synthesis of a series of new
degradable multivalent cationic lipids, termed CMVLn (n=2 to 5).
These CMVLs contain a reductively cleavable disulfide bond in the
spacer between the hydrophobic moiety and the cationic head group.
Reducing conditions result in separation of the hydrophobic tail from
the headgroup. Except for the spacer, the CMVLs are identical to a
previously synthesized series of multivalent lipids [32]. Of these,
MVL5 in particular is a highly efficient vector for both DNA and siRNA
[26,27,32]. The headgroup charge of the CMVLs was measured using
an ethidium bromide (EtBr) displacement assay. CL–DNA complexes
prepared from mixtures of the CMVLs with neutral 1,2-dioleoyl-sn-
glycero-3-phosphatidylcholine (DOPC) were studied using small-
angle X-ray scattering (SAXS) under reducing and non-reducing
conditions, as well as measurements of cytotoxicity and TE. The
CMVLs were compared with commercially available 2,3-dioleoylox-
ypropyltrimethylammonium chloride (DOTAP) and Lipofectamine
2000 as well as with non-degradable MVL5. All CMVL/DOPC–DNA
complexes form lamellar phases (LαC) which can be completely
disassembled by addition of reducing agents such as GSH and DTT.
Importantly, the complexes of CMVLn (n=3, 4, 5) exhibit high TE at
reduced cytotoxity when compared to non-degradable analogs and
commercial lipid reagents.
2.3. Lipid suspensions
Lipid stock solutions in chloroform (for DOTAP and DOPC) or
chloroform/methanol (9:1, v/v; for MVL5 and all CMVLs) were
combined at the appropriate ratios in glass vials. The obtained lipid
solutions were dried, first by a stream of nitrogen and subsequently in
a vacuum for 12 h, to form a thin lipid film. Sterile high resistivity
(18.2 MΩ cm) water was added and the mixture was incubated at
37 °C for at least 12 h to give suspensions of a final concentration of
50 mM for X-ray samples. For transfection, aqueous lipid suspensions
were prepared at 0.5 mM. Lipid mixtures containing higher mole
fractions of DOPC formed opaque suspensions which were tip-
sonicated to clarity (Vibra-cell, Sonics Materials). The aqueous lipid
suspensions were stored at 4 °C until use.
2.4. Ethidium bromide displacement assay
Wells in a 96-well plate were preloaded with a mixture of 2.4 μg
of DNA and 0.28 μg of EtBr in 100 μL of water. To the wells, varied
amounts of a 0.2 mM aqueous lipid solution (at a lipid mole fraction of
DOPC (ΦDOPC)=0) were added, preparing at least duplicate samples
for each data point. Fluorescence was measured on a 1420 Multilabel
counter Victor 3 V (Perkin Elmer) plate reader. The results were
normalized to the fluorescence reading in the absence of lipid. To
calculate the lipid headgroup charge, Z, the following equation
was used: at the isoelectric point (determined from the data as
Nþ
N−
mCL = MCL
described [32]),
= 1 =
Z2, where N+ and N− are the
number of cationic charges omnDNtAh=eMliDpNAiꢀdbpand of negative charges on the
DNA, respectively; mCL and mDNA are the mass of cationic lipid and of
DNA respectively; and MCL and MDNA-bp are the molecular weight of
cationic lipid and DNA basepair, respectively.
2.5. Optical microscopy
For imaging of complexes with optical microscopy, 0.2 wt.%
(of total lipid) DHPE-Texas Red (Avanti Polar Lipids) was added to
the lipid mixture (before evaporation of the organic solvent). This
fluorescent dye has an excitation/emission peak at 585/615 nm. Cy5
(Mirus Bio LLC), with an excitation/emission peak at 605/670 nm, was
conjugated to DNA using the manufacturer's protocol. Complexes
were then prepared as for transfection and imaged in Opti-MEM,
20 min after complex formation. Fluorescence intensity profiles of
particles were obtained with the ImageJ Color Profiler software tool.
2. Materials and methods
2.1. Materials and reagents
2.6. Dynamic light scattering (DLS)
MVL5 was prepared as described [26]. Opti-MEM cell culture
medium and Lipofectamine 2000 were purchased from Invitrogen.
The lipids DOTAP and DOPC were purchased from Avanti Polar Lipids.
The chemical structures of MVL5, DOTAP and DOPC are shown in the
Complexes for DLS were prepared as for transfection but at a final
concentration of 2 μg/mL of DNA (twice the concentration used for
transfection, to achieve a sufficient particle concentration for the DLS

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measurement). The complexes were analyzed by DLS in Opti-MEM,
20 min after complex formation using a Malvern Nano ZS (173°
scattering angle). The plot shows the z-average hydrodynamic
diameter as obtained from the instrument. Data points shown are
the average of duplicate measurements of the same sample, with
error bars showing the standard deviation.
2.10. Synthesis
2.10.1. General procedure for the deprotection of Boc-protected lipids
The protected lipid (0.05–0.25 g) was cooled on an ice bath under
a nitrogen atmosphere and dissolved with stirring in cooled
trifluoroacetic acid (10 mL/g lipid; from Fisher Scientific) that had
been saturated with nitrogen gas. The solution was stirred in a water
bath at room temperature for 30 min and all volatiles evaporated in
vacuo overnight.
2.7. Small-angle X-ray scattering
High-resolution SAXS experiments were performed at the Stanford
Synchrotron Radiation Lightsource. X-ray samples were prepared from
50 mM liposome solutions and 4 mg/mL DNA solutions, using 100 μg of
DNA. CL–DNA complexes were formed by combining the CL and DNA
solutions in microcentrifuge tubes, diluting 1-fold with Opti-MEM and
incubating at 4 °C for at least 3 days after extensive centrifugation. For
the samples containing reducing agents, GSH and DTT stock solutions
were prepared at 300 mM and added to each sample after complex
formation (after centrifugation) at a molar ratio of ≥10 over the CMVLs.
Samples were then transferred into 1.5 mm quartz capillaries (Hilgen-
berg) and flame-sealed. Typically, the CL–DNA complexes formed a
white opaque precipitate. Samples containing reducing agents occa-
sionally exhibited phase separation of the initial precipitate. SAXS
measurements were performed at multiple positions throughout the
samples and representative scans are shown.
2.10.2. Oleyl alcohol (1)
To a solution of 12.5 g (44.3 mmol) oleic acid (Alfa Aesar) in
150 mL of ether was added with stirring a mixture of 2.15 g
(56.7 mmol) lithium aluminum hydride and 30 mL of THF in portions.
When refluxing had subsided, the mixture was heated to reflux for
45 min. Excess lithium aluminum hydride was hydrolyzed by the
addition of wet ether, then water, and finally 2 M hydrochloric acid.
The resulting mixture was acidified with conc. hydrochloric acid and
the phases separated. The aqueous phase was extracted twice with
CH2Cl2, and the combined organic phases were washed twice with
water, dried (Na2SO4), and evaporated in vacuo to yield 11.7 g
(43.4 mmol, 98%) of oleyl alcohol as a colorless liquid: Rf =0.23
(cyclohexane/ethylacetate 4:1), identical to a commercial sample.
To assess the stability of CMVL-containing CL–DNA complexes (cf.
Supplementary data), each X-ray sample was measured at least twice
(unless the sample capillary was inadvertently broken during
handling). The second measurements were performed (on the same
sealed sample, but at a different location within the capillary) no
earlier than one month later. Repeat experiments (of independently
prepared samples), performed for a number of lipid compositions of
CMVL5, resulted in scans that matched the original ones.
2.10.3. 3,4-Di(oleyloxy)benzoic acid (DOB; 2)
Under cooling with an ice/water bath, 4.97 g (27.3 mmol) 3,4-
dihydroxy-benzoic acid ethyl ester (TCI America) was added to a
solution of 21.49 g (81.94 mmol) of triphenylphosphine in 246 mL of
THF. After stirring for 30 min, a total of 37.3 mL of a 40 wt,% solution of
DEAD (81.9 mmol) and a solution of 22.0 g (81.9 mmol) of 1 in 41 mL
of toluene were added dropwise (using two separate addition
funnels), maintaining an internal temperature of less than 5 °C.
After stirring for 18 h at room temperature, the reaction mixture was
heated to 50 °C, and stirring was continued for another 3 h. The
solvents were evaporated and the residue suspended in 44 mL of
ether by stirring for 10 min. A total of 22 mL of hexane was added and
the mixture was incubated for 15 min with sonication (bath
sonicator), filtered, and the residue washed with 110 mL ether/
hexane (1:1, v/v). The combined filtrates were evaporated in vacuo, a
solution of 7.69 g (137 mmol) of KOH in 210 mL ethanol was added
and the mixture was heated to reflux for 4 h under nitrogen. The hot
reaction mixture was added to 260 mL of water and filtered. The
residue was washed three times with water and recrystallized from
75 mL of ethanol to yield 9.8 g (14.34 mmol, 52.5%) of DOB [53] as a
colorless solid: Rf =0.26 (cyclohexane/ethylacetate 4:1), NMR spectra
identical to a genuine sample [53].
2.8. Cytotoxicity
Cytotoxicity was measured using the CellTiter 96 AQueous One
Solution Cell Proliferation Assay (Promega). Mouse fibroblast L-cells
were seeded in 96-well plates at approximately 15000 cells per well.
After 18 h of incubation, the cells were treated with CL–DNA complexes
in Opti-MEM using a total of 0.08 μg DNA per well. After 6 h incubation,
the CL–DNA complex solution was replaced by a mixture of 100 μL of
Opti-MEM and 20 μL of the Cell Proliferation Assay, followed by 2 h of
incubation. Cytotoxicity was then determined by colorimetric mea-
surement on a 1420 Multilabel counter Victor 3 V (Perkin Elmer) plate
reader as per the assay manufacturer's instructions. The experiment
was performed simultaneously for all lipids and the results were
normalized to control wells, which differed from the experimental
wells only in that they were treated with Opti-MEM instead of
complexes. All data points were measured as quadruplicates.
2.10.4. [2-(2-Amino-ethyldisulfanyl)-ethyl]-carbamic acid tert-butyl
ester (3)
To a solution of 8.0 g (36 mmol) cystamine bis hydrochloride in
200 mL methanol were added 14.9 mL (107 mmol) of triethylamine
and 7.7 g (36 mmol) of di-tert-butyldicarbonate (Fluka). The mixture
was stirred for 30 min at room temperature and the solvent
evaporated in vacuo. To the residue, 80 mL of 1 M aq NaH2PO4
(pH=4.2) was added and the mixture was extracted twice with ether
to remove di-Boc-cystamine. The aqueous solution was basified
(pH=9) with 1 M NaOH and extracted twice with EtOAc. The
combined EtOAc phases were washed twice with water, dried
(Na2SO4) and evaporated in vacuo to yield 4.10 g (16.2 mmol, 45%)
of 3 as a white solid: Rf =0.47 (CHCl3/MeOH 9:1, 1% concentrated
NH4OH); NMR spectra matching literature data [54].
2.9. Cell transfection
Mouse fibroblast L-cells were cultured and transfected as previously
reported [52]. Briefly, cells in 24-well plates were incubated for 6 h with
CL–DNA complexes containing a constant 0.4 μg of plasmid DNA,
washed with phosphate-buffered saline, and the complex-containing
medium replaced by serum-containing culture medium. For samples
containing Lipofectamine 2000, 1 μg reagent per sample was used. Cells
were then incubated for 18 h before luciferase expression was
measured using the luciferase assay (Promega) as per manufacturer's
instructions. Light output readings were measured as relative light
units (RLU) on a 1420 Multilabel counter Victor 3 V (Perkin Elmer)
plate reader. These readings were normalized to the weight of total
cellular protein determined using Bio-Rad Protein Assay Dye Reagent.
Data points shown in the same plot were measured on the same day,
and all data points were measured in duplicate.
2.10.5. [2-(2-{[3,4-Di(oleyloxy)benzoyl]amino}-ethyldisulfanyl)ethyl]-
carbamic acid tert-butyl ester (4)
Under a nitrogen atmosphere, 91 mL (5.4 mmol) of N,N-diisopro-
pylethylamine (DIEA) were added with stirring to a mixture of 0.971 g

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2159
(3.03 mmol) of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) and 1.771 g (2.703 mmol) of 2 in CH2Cl2. After
20 min, 0.761 g (3.03 mmol) of 3 in CH2Cl2 was added and stirring was
continued for 18 h. The reaction mixture was extracted twice with 1 M
aq NaH2PO4, twice with water, twice with 1 M aq NaHCO3, and twice
more with water, dried (Na2SO4), and evaporated in vacuo to yield
2.02 g (2.27 mmol, 84%) of 4 as an off-white solid: Rf=0.26 (CHCl3/
MeOH 100:1); 1H NMR (CDCl3, 200 MHz): δ=0.87 (“t”, J=6.4 Hz, 6H,
CH2CH3), 1.1–2.2 (several m, lg. peaks @ 1.27, 1.31, 1.44, 1.82 (bm),
2.00, 2.02, 65H, C―CH2―C, C(CH3)3), 2.80 (t, J=6.5 Hz, 2H, S―CH2),
2.90 (t, J=6.0 Hz, 2H, S―CH2), 3.45, 3.74 (2 “q”, J≈6.1 Hz, 4H,
N―CH2), 4.03 (“t”, J=5.6 Hz, 4H, O―CH2), 5.08 (b, 1H, NH), 5.3–5.6
(m, 4H, CH), 6.87 (d, 3J=8.4 Hz, 1H, Harm-C(O)), 6.97 (b, 1H, NH),
7.35–7.5 (m, 2H, Har); 13C NMR (125 MHz, CDCl3): δ=14.2 (CH2CH3),
22.8, 26.1, 27.3, 29.4, 29.6, 29.9, 32.0 (C―CH2―C), 28.5 ((CH3)3C), 38.4
(b), 39.1, 39.6 (S―CH2, CH2―N), 69.1, 69.3 (CH2―O), 79.4 ((CH3)3C),
112.4, 113.0, 119.8 (CarH), 126.9 (Car―C(O)), 129.9, 130.0 ( CH), 149.1,
152.1 (Car―O), 154.2 (C(O)O), 167.5 (Car―C(O)).
1.66, 1.87, 60H, C―CH2―C), 2.65–2.85 (2 m, 6H, S―CH2, CH2―N),
3.35–3.6, (2 m, 4H, CH2―N), 3.7–3.85 (m, 1H, CH), 3.85–3.95 (m, 4H,
O―CH2), 5.1–5.3 (m, 4H, CH), 6.74 (d, 1H,3J=8.3 Hz, Harm-C(O)),
7.15–7.3 (m, 2H, Har); 13C NMR (125 MHz, CDCl3/MeOH-d4 9:1):
δ=13.8 (CH3), 22.5, 25.89, 25.93, 27.0, 28.0, 29.02, 29.12, 29.2, 29.26,
29.29, 29.7, 31.8 (C―CH2―C), 37.1, 37.2, 38.3, 38.5, 39.1 (S―CH2,
N―CH2), 52.3 (N―CH), 69.0, 69.5 (CH2―O), 112.3, 112.9, 120.4
(Car―H), 116.3 (q, J=299 Hz , F3C), 126.2 (Car―CO), 129.7, 129.8
( CH―CH2), 148.6, 152.2 (Car―O), 161.6 (q, J=35.5 Hz , F3C―C), 168.5,
168.6(C(O)―N).
2.10.9. {(S)-1-[2-(2-{(3,4-Di(oleyloxy)benzoyl)amino}-ethyldisulfanyl)-
ethylcarbamoyl]-4-tert-butoxycarbonylamino-butyl}-(3-tert-
butoxycarbonylamino-propyl)-carbamic acid tert-butyl ester (BocCMVL3)
To a mixture of 0.38 g (0.78 mmol) of (S)-2-(tert-butoxycarbonyl(3-
(tert-butoxycarbonylamino)propyl)amino)-5-(tert-butoxycarbonyla-
mino)pentanoic acid and 0.27 g (0.85 mmol) of TBTU in CH2Cl2 were
added 203 μL (1.16 mmol) of DIEA with stirring. After 10 min, the
resulting solution was added to a mixture of 0.77 g (0.85 mmol) of 5
and 203 μL (1.16 mmol) of DIEA in CH2Cl2 and stirring was continued
for 18 h. The reaction mixture was extracted twice with 1 M aq
NaH2PO4, once with water, twice with 5% NaHCO3, and once more with
water. The organic phase was dried (Na2SO4), evaporated in vacuo and
the residue purified by flash chromatography on 85 g of silica gel using
CHCl3/MeOH (100:1) as the eluent to yield 0.747 g (0.580 mmol, 72%)
of BocCMVL3 as a colorless solid: Rf=0.41 (CHCl3/MeOH 20:1); 1H
NMR (500 MHz, CDCl3/MeOH-d4 9:1) δ=0.72 (“t”, J=6.0 Hz, 6H,
CH2CH3), 0.9–2.0 (several m, 89H, lg. peaks @ 1.12, 1.17, 1.27, 1.31, 1.53
(b), 1.68 (b), 1.87, C―CH2―C, C(CH3)3), 2.6–2.7 (2H), 2.7–2.85 (2H),
2.85–3.0 (4H), 3.0–3.15 (2H) (4 bm, S―CH2, N―CH2), 3.35–3.45 (bm,
2H), 3.5–3.6 (bm, 2H) (CH2―N), 3.8–3.95 (m, 4H, O―CH2), 4.23 (b, 1H,
CH), 5.3–5.7 (m, 4H, CH), 5.4 (b, 1H, NH), 6.73 (“d”, 3J=8.2 Hz, 1H,
Harm-C(O)), 7.2–7.3 (m, 2H, CarH), 7.53 (b, 1H, NH); 13C NMR
(125 MHz, CDCl3/MeOH-d4 9:1): δ=13.9 (CH2CH3), 22.5, 25.90,
25.94, 27.1, 29.1, 29.19, 29.23, 29.30, 29.34, 29.4, 29.5, 29.6, 31.8
(C―CH2―C), 28.09, 28.12 ((CH3)3C), 37.2, 37.6, 38.0 (b), 38.1 (b), 39.0,
39.7 (CH2―N, S―CH2), 58.5 (CH―N), 69.1, 69.4 (CH2―O), 79.1, 81.1
((CH3)3C), 112.5, 113.0, 120.3 (CarH), 126.4 (Car―CO), 129.8, 129.9
( CH2), 148.7, 152.0 (Car―O), 156.60, 156.64 (C(O)O), 168.0, 171.9
(C(O)―N).
2.10.6. N-[2-(2-Amino-ethyldisulfanyl)-ethyl]-3,4-di(oleyloxy)benzamide
trifluoroacetate (5)
As described in the general procedure, 1.9 g (2.1 mmol) of 4 were
deprotected to yield 1.76 g (1.95 mmol, 91%) of 5 as an off-white solid:
Rf=0.3 (CHCl3/MeOH 9:1); 1H NMR (200 MHz, CDCl3): δ=0.91 (“t”,
J=6.4 Hz, 6H, CH3), 1.0–2.2 (m, lg. peaks at 1.25, 1.80 (b), 2.04, 2.06,
56H, C―CH2―C), 2.7–3.1 (b, 4H, S―CH2), 3.2–3.5, 3.5–3.8 (2 b, 4H,
N―CH2), 3.8–4.2 (m, 4H, O―CH2), 5.0–5.25 (m, 5H, CH―N), 5.25–5.55
(m, 4H, CH), 6.83 (d, 3J=8.1 Hz, 1H, Harm-C(O)), 7.2–7.5 (m, 2H, Har),
8.2 (b, 4H, NH); 13C NMR (50 MHz, CDCl3): δ=14.2 (CH2CH3), 22.8,
26.2, 27.4, 29.5, 29.7, 29.9, 32.0 (C―CH2―C), 37.3 (2 C), 39.2, 39.6
(S―CH2, CH2―N), 69.3, 69.6 (CH2―O), 112.4, 112.8, 120.7 (CarH), 126.2
(Car―C(O)), 129.9, 130.0 ( CH), 149.1, 152.5 (Car―O), 168.2 (C(O)N).
2.10.7. {(S)-1-[2-(2-{(3,4-Di(oleyloxy)benzoyl)amino}-ethyldisulfanyl)-
ethylcarbamoyl]-4-tert-butoxycarbonylamino-butyl}-carbamic acid tert-
butyl ester (BocCMVL2)
To a mixture of 0.54 g (1.61 mmol) of Nα,Nδ-Bis(Boc)-ornithine [28]
and 0.57 g (1.8 mmol) of TBTU in CH2Cl2 were added 422 μL
(2.42 mmol) of DIEA with stirring. After 10 min, the resulting solution
was added to a mixture of 1.605 g (1.78 mmol) of 5 and 422 μL
(2.42 mmol) of DIEA. After stirring for 18 h, the reaction mixture was
extracted three times with 1 M aq NaH2PO4, twice with water, twice
with 5% NaHCO3 and twice more with water. The organic phase was
dried (Na2SO4) and evaporated in vacuo and the residue purified by
flash chromatography on 150 g of silica gel using CHCl3/MeOH (100:1,
v/v) as the eluent to yield 1.502 g (1.361 mmol, 84%) of BocCMVL2 as a
colorless solid: Rf=0.28 (CHCl3/MeOH 40:1); 1H NMR (200 MHz,
CDCl3): δ=0.81 (“t”, J=6.3 Hz, 6H, CH2CH3), 0.94–2.1 (several m, lg.
peaks @ 1.20, 1.25, 1.36, 1.75 (b), 1.93, 1.96, 78H, C―CH2―C, C(CH3)3),
2.7–3.4, 3.4–3.7 (several m, 6+4H, S―CH2, N―CH2), 3.8–4.1 (m, 4H,
O―CH2), 4.1–4.3, 4.5–4.8 (2 bm, 2H, NH), 4.8–5.1 (m, 1H, CH), 5.1–5.4
(m, 4H, CH―), 6.79 (d, 3J=8.4 Hz, 1H, Harm-C(O)), 6.89, 7.11 (2 bt, 2H,
NH), 7.3–7.5 (m, 2H, Har); 13C NMR (50 MHz, CDCl3): δ=14.0
(CH2CH3), 22.5, 25.9, 26.2, 27.1, 29.0, 29.1, 29.2, 29.26, 29.29, 29.4,
29.6, 31.8, 33.6 (C―CH2―C), 28.2, 28.3 ((CH3)3C), 37.6, 37.9, 38.2, 39.1
(S―CH2, CH2―N), 53.2 (CH), 69.0, 69.3 (CH2―O), 79.1, 79.7 (C(CH3)3),
112.2, 112.8, 119.8 (CarH), 126.4 (Car―C(O)), 129.6, 129.8 ( CH), 148.8,
152.0 (Car―O), 155.7, 156.3 (C(O)O), 167.4, 172.6 (C(O)N).
2.10.10. N-(2-((2-(5-Amino-(2S)-2-(3-aminopropylamino)pentanamido)
ethyl)disulfanyl) ethyl)-{3,4-di(oleyloxy)benzamide} tri-trifluoroacetate
(CMVL3)
As described in the general procedure, 225 mg (175 μmol) of
BocCMVL3 were deprotected to yield 220 mg (165 μmol, 94%) of
CMVL3 as a colorless solid: Rf=0.46 (CHCl3/MeOH/NH4OH 6:3:1); 1H
NMR (500 MHz, CDCl3/MeOH-d4 9:1): δ=0.77 (“t”, 6H, J=6.8 Hz,
CH2CH3), 1.0–2.1 (several m, 62H, lg. peaks at 1.16, 1.21, 1.35 (m), 1.67
(m), 1.86 (m), C―CH2―C), 2.76 (t, J=6.3 Hz), 2.81 (t, J=6.8 Hz), 2.85
(t, J=7.0 Hz), 2.87–3.1 (2 m), (12H, S―CH2, N―CH2), 3.6–3.75 (2 m,
4H, CH2―N), 3.8–4.0 (m, 4H, O―CH2), 4.85–5.15 (m, 1H, CH), 5.15–5.3
(m, 4H, CH), 6.78 (d, 3J=8.5 Hz, 1H, Harm-C(O)), 7.2–7.3 (m, 2H,
Har);13C NMR (125 MHz, CDCl3/MeOH-d4 9:1): δ=13.9 (CH3), 22.4,
22.6, 24.9, 26.0, 27.0, 27.1, 27.2, 29.1, 29.2, 29.25, 29.29, 29.4, 29.5, 30.7,
31.7, 31.8, 31.9, 33.6 (C―CH2―C), 36.5, 37.23, 37.27, 38.4, 38.6, 39.0,
43.4 (S―CH2, CH2―N), 59.6 (CH―N), 69.1, 69.5 (CH2―O), 112.4, 113.0,
120.4 (CarH), 126.3 (Car―CO), 129.8, 129.9 ( CH), 148.7, 152.3 (Car―O),
167.3, 168.3 (C(O)N).
2.10.8. N-(2-((2-((2S)-2,5-Diaminopentanamido)ethyl)disulfanyl)
ethyl)-{3,4-di(oleyloxy) benzamide} di-trifluoroacetate (CMVL2)
As described in the general procedure, 50 mg (45 μmol) of
BocCMVL2 were deprotected using 800 μL of TFA to yield 50 mg
(45 μmol, 98%) of CMVL2 as a colorless solid: Rf=0.33 (CHCl3/MeOH/
NH4OH 12:4:1). 1H NMR (500 MHz, CDCl3/MeOH-d4 9:1): δ=0.72 (“t”,
J=5.8 Hz, 6H, CH3), 0.9–2.0 (several m, lg. peaks @ 1.12, 1.17, 1.32 (b),
2.10.11. {(S)-1-[2-(2-{(3,4-Di(oleyloxy)benzoyl)amino}-ethyldisulfanyl)-
ethylcarbamoyl]-4-[tert-butoxycarbonyl-(3-tert-butoxycarbonylamino-
propyl)-amino]-butyl}-(3-tert-butoxycarbonylamino-propyl)-carbamic
acid tert-butyl ester (BocCMVL4)
To a mixture of 0.46 g (0.71 mmol) of Nα,Nδ-Bis(Boc)-Nα,Nδ-bis(3-
[Boc-amino]propyl)ornithine [52] and 0.25 g (0.78 mmol) TBTU in

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R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
CH2Cl2 were added 184 μL (1.06 mmol) of DIEA with stirring. After
10 min, the resulting solution was added to a mixture of 0.70 g
(0.78 mmol) of 5 and 184 μL (1.06 mmol) of DIEA in CH2Cl2 and
stirring was continued for 18 h. The reaction mixture was extracted
twice with 1 M aq NaH2PO4, once with water, twice with 5% NaHCO3,
and once more with water. The organic phase was dried (Na2SO4),
evaporated in vacuo and the residue purified by flash chromatography
on 85 g of silica gel using CHCl3/MeOH (100:1) as the eluent to yield
0.720 g (0.507 mmol, 70%) of BocCMVL4 as a colorless solid: Rf =0.26
(CHCl3/MeOH 40:1); 1H NMR (500 MHz, MeOH-d4): δ=0.89 (“t”, 6H,
J=6.4 Hz, CH2―CH3), 1.1–2.2 (several m, lg. peaks @ 1.28, 1.34, 1.40,
1.43, 1.45, 1.47, 1.69 (b), 1.79 (b), 2.03 (b), 100H, C―CH2―C, C
(CH3)3), 2.86 (t, J=6.0 Hz, 2H), 2.95 (t, J=6.4 Hz, 2H), 3.03 (t,
J=6.0 Hz, 4H), 3.1–3.4 (m, 6H+MeOH-d3), 3.52 (t, J=6.0 Hz, 2H),
3.67 (t, J=6.6 Hz, 2H) (S―CH2, CH2―N), 4.0–4.1 (m, 4H, O―CH2),
4.52 (b, 1H, CH), 5.3–5.45 (m, 4H, CH), 6.94 (d, 3J=8.8 Hz, 1H, Harm-
C(O)), 7.45 (b, 2H, Har); 13C NMR (125 MHz, MeOH-d4): δ=14.8
(CH2CH3), 23.9, 27.5, 28.4, 30.5, 30.6, 30.66, 30.73, 30.8, 30.9, 31.1,
33.3 (C―CH2―C), 29.0, 29.08, 29.12 ((CH3)3CO), 38.6, 38.8, 39.0 (b),
39.3 (b), 39.7 (b), 40.6 (S―CH2, CH2―N), 70.3, 70.7 (C―CH2―O), 80.1
(b), 81.2, 82.0 (b) ((CH3)3C), 113.9, 114.5, 122.2 (CarH), 127.9 (Car―C
(O)), 131.0, 131.1 ( CH), 150.3, 153.8 (Car―O), 157.5, 158.5 (2 b, C(O)
O), 169.8, 173.7 (b) (C(O)N).
(125 MHz, CDCl3): δ=14.6 (CH2CH3), 22.8, 26.2, 27.4, 29.3, 29.5,
29.6, 29.7, 30.0, 32.1 (C―CH2―C), 28.7 ((CH3)3C), 37.7, 38.1, 38.4, 39.1,
51.8, 53.4 (S―CH2, CH2―N), 53.4 (CH―N), 69.3, 69.5 (C―CH2―O),
79.2 (b), 81.2 ((CH3)3C), 112.5, 113.2, 119.9 (CarH), 126.9 (Car―C(O)),
130.0, 130.1 ( CH), 149.1, 152.2 (Car―O), 156.28, 156.31 (C(O)O), 167.5,
171.9 (C(O)N).
2.10.14. N-(2-((2-((2S)-2-(3-Aminopropylamino)-5-(bis(3-
aminopropyl)amino) pentanamido)ethyl)disulfanyl)ethyl)-{3,4-di
(oleyloxy)benzamide} penta-trifluoroacetate (CMVL5)
As described in the general procedure, 63 mg (43 μmol) of
BocCMVL5 were deprotected using 2 mL of TFA. The obtained residue
was dissolved in 100 μL methanol, 20 mL of diethylether/pentane (1:1,
v/v) were added and the mixture was incubated for 16 h at 4 °C. The
resulting precipitate was isolated by decanting, washed twice with
5 mL diethylether/pentane (1:1, v/v) and dried to yield 67 mg
(41 μmol, 95%) of CMVL5 as a colorless solid: Rf =0.32 (CHCl3/
MeOH/25% NH4OH 40:24:10); 1H NMR (500 MHz, MeOH-d4):
δ=0.82 (b, 6H, CH2CH3), 1.01–2.49 (several m, 66H, lg. peaks at 1.22,
1.28, 1.45, 1.60, 1.74, 1.86, 1.97, 2.09 C―CH2―C), 2.75–3.35 (22H,
S―CH2, N―CH2), 3.42–3.72 (m, 4H, O―CH2), 3.8–4.1 (b, 5H, NH, CH),
4.92–5.2 (b, 4H, CH), 6.9 (d, 3J=5 Hz, 1H, Harm-C(O)), 7.2–7.6 (m, 2H,
Har);13C NMR (125 MHz, MeOH-d4 9): δ=14.6 (CH3), 20.7, 23.3, 23.9,
25.5, 26.2, 26.3, 27.5, 28.3, 28.7, 30.47, 30.52, 3.57, 30.6, 30.7, 30.8, 31.0,
31.05 (C―CH2―C), 33.2, 33.8, 34.9, 38.0, 38.5, 39.4, 40.5, 45.2, 51.4,
53.7 (S―CH2, CH2―N), 61.5 (CH), 70.3, 70.8 (CH2―O), 113.9, 114.4,
122.3 (CarH), 127.8 (Car―C(O)), 131.0, 131.04 ( CH), 150.3, 154.0
(Car―O―CH2), 163.0 (q, J=34.8 Hz, F3C―C(O)), 168.7, 170.1 (C(O)N).
2.10.12. N-(2-(((2S)-2-(2,5-Bis(3-aminopropylamino)pentanamido)
ethyl)disulfanyl) ethyl)-{3,4-di(oleyloxy)benzamide} tetra-trifluoroacetate
(CMVL4)
As described in the general procedure, 232 mg (164 μmol) of
BocCMVL4 were deprotected to yield 238 mg (161 μmol, 98%) of
CMVL4 as a colorless solid: Rf =0.41 (CHCl3/MeOH/25% NH4OH
6:2:1). 1H NMR (500 MHz, MeOH-d4): δ=0.81 (“t”, 6H, J=7 Hz,
CH2CH3), 1.1–2.1 (several m, 64H, lg. peaks at 1.21, 1.27, 1.37, 1.44
(m), 1.59 (b), 1.73 (m), 1.95, 2.00 (m) C―CH2―C), 2.78–2.92 (m, 4H,
S―CH2), 2.92–3.15 (m, 10H, N―CH2), 3.42–3.68 (m, 4H, HN―CH2),
3.87 (t, 1H, J=6.3, +H2N―CH), 3.9–4.02 (m, 4H, O―CH2), 5.2–5.35
(m, 4H, CH), 6.90 (d, 3J=8.1 Hz, 1H, Harm-C(O)), 7.4–7.5 (m, 2H, Har),
8.0 (b, NH); 13C NMR (125 MHz, MeOH-d4): δ=14.6 (CH3), 22.9, 23.9,
25.48, 25.53, 26.3, 27.5, 28.3, 28.7, 30.7, 30.78, 30.82, 30.9, 30.94,
30.97, 31.0, 31.04, 33.2, 34.9 (C―CH2―C), 38.0, 38.52, 38.55, 39.69,
40.54, 45.1, 46.0, 48.3 (CH2―N, S―CH2), 61.5 (CH), 70.3, 70.8
(CH2―O), 113.9, 114.4, 122.3 (CarH), 118.0 (q, J=290 Hz, CF), 127.8
(Car―C(O)), 130.9, 131.0 ( CH), 150.3, 154.0 (Car―O), 162.6 (q,
J=34.2 Hz, F3C―C(O)), 168.6, 170.1 (C(O)).
3. Results and discussion
3.1. Lipid design and synthesis
The CMVLn (n=2 to 5) lipids presented in this study were
designed to be stable in the extracellular environment but to readily
degrade in the reducing environment of the cytoplasm. We
anticipated that this would reduce lipid toxicity and enhance nucleic
acid release from their DNA complexes. The former is of practical
importance (in particular for siRNA delivery [2]) while the latter
should shed light on the delivery pathway of multivalent lipids,
particularly the limitations observed at high membrane charge
density (σM) [32]. As evident from their chemical structures shown
in Table 1, the CMVLs are closely related to the MVL series of lipids
previously synthesized in our group [26,32,55]: MVLs and CMVLs
differ only in the spacer connecting lipid tails and headgroup, which is
slightly longer and incorporates a reductively degradable disulfide
bond in the CMVLs. The synthesis of the CMVLs made use of the
headgroup building blocks developed for the synthesis of the MVLs
[26,28] as well as the same hydrophobic moiety (3,4-di(oleyloxy)
benzoic acid). However, a new synthesis of DOB was devised to
circumvent the use of expensive oleyl bromide as the starting
material. Thus, oleyl alcohol was synthesized by reduction of oleic
acid and used to alkylate ethyl(3,4-(dihydroxy))benzoate in a
Mitsunobu reaction [56,57] to yield DOB (2) after saponification (cf.
Fig. 1). The synthesis proceeded by coupling DOB with mono-Boc-
protected cystamine 3 [54] and Boc-deprotection to yield 5, the
common precursor for the CMVLs. As exemplified for CMVL4 in Fig. 1,
precursor 5 was coupled with the Boc-protected headgroup building
blocks, and the resulting Boc-protected CMVLs were purified
extensively and finally deprotected with TFA.
2.10.13. {4-[Bis-(3-tert-butoxycarbonylamino-propyl)amino](S)-1-[2-
(2-{(3,4-di(oleyloxy)benzoyl)amino}-ethyldisulfanyl)-ethylcarbamoyl]-
butyl}-(3-tert-butoxycarbonylamino-propyl)-carbamic acid tert-butyl
ester (BocCMVL5)
To a mixture of 575 mg (817 μmol) of Nα-Boc-Nα,Nδ,Nδ-tris(3-[Boc-
amino]propyl)ornithine [26] and 289 mg (899 μmol) of TBTU in CH2Cl2
were added 116 μL (899 μmol) of DIEA with stirring. After 10 min, the
resulting solution was added to a mixture of 812 mg (899 μmol) of 5
and 116 μL (899 μmol) of DIEA in CH2Cl2 and stirring was continued for
18 h. The reaction mixture was extracted twice with 1 M aq NaH2PO4,
once with water, twice with 5% NaHCO3, and once more with water.
The organic phase was dried (Na2SO4), evaporated in vacuo and the
residue purified by flash chromatography on 120 g of silica gel using
CHCl3/MeOH (100:1) as the eluent to yield 643 mg (436 μmol, 52%) of
BocCMVL5 as a colorless solid: Rf=0.32 (CHCl3/MeOH 9:1); 1H NMR
(200 MHz, CDCl3) δ=0.75 (“t”, J=6.4 Hz, 6H, CH2―CH3), 0.9–2.3
(several m, lg. peaks @ 1.26, 1.31, 1.43, 1.47; 102H, C―CH2―C, C
(CH3)3), 2.46 (b, 6H), 2.82 (t, 2H), 2.93 (t, 2H), 3.0–3.35 (m, 8H) 3.45–
3.85 (2 “q”, 4H) (S―CH2, N―CH2), 3.9–4.15 (m, 4H, O―CH2), 4.37 (b,
1H), 4.95 (b, 1H), 5.1–5.5 (m, 5H) (NH, N―CH, CH), 6.84 (d,3J=8.4 Hz,
1H, Harm-C(O)), 7.00 (b, 1H, NH), 7.34 (dd, 3J=8.4 Hz, 4J=1.9 Hz, 1H,
CL–DNA complexes were prepared from mixtures of the CMVLs
and DOPC at varied mole fractions of CMVL (ΦCMVL =1−ΦDOPC) and
using varying amounts of DNA. The complexes were characterized
using small-angle X-ray scattering, optical microscopy, and dynamic
light scattering, and their cytotoxicity and transfection efficiency were
assessed and compared to both MVL5 (a highly efficient, non-
H
arp-OR), 7.44 (d, 4J=1.9 Hz, 1H, Haro-C(O), o-OR); 13C NMR

R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
2161
Table 1
Chemical structures, abbreviated names, and headgroup charges of the synthesized degradable cationic lipids.
Name
CMVL2
CMVL3
CMVL4
CMVL5
Headgroup structurea
Chargea
+2
+3
+4
+5
a
At full protonation.
degradable analog of CMVL5) [26,27,32] and commercially available
transfection agents (DOTAP and Lipofectamine 2000). The chemical
structures of MVL5, DOTAP and DOPC are shown in the Supplemen-
tary data, Fig. S1.
3.4. Small-angle X-ray scattering
We used SAXS to probe the structures of CMVL/DOPC–DNA
complexes in the absence and presence of reducing agents. No signs
of lipid phase separation could be detected; hence the complexes
contained both CMVL and DOPC at the nominal ratios. Fig. 5 shows
SAXS scans obtained for CMVL2 and CMVL5 at ΦCMVL =0.6 with and
without reducing agents (DTT and GSH). CMVL/DOPC–DNA com-
plexes prepared in non-reducing environments are highly stable as
judged by SAXS scans. As shown in the Supplementary data, the
lamellar structure shows no signs of disintegration after 6 months and
(for CMVL5, the only lipid investigated over such a long time period)
predominates even after 41 months. The scattering patterns of these
3.2. Determination of the headgroup charge
An EtBr displacement assay [58,59] was used to estimate the head
group charge of the cationic lipids as reported previously [32]. EtBr
fluoresces when intercalated into DNA but self-quenches in solution.
Therefore, the fluorescence of a mixture of DNA and EtBr decreases
when cationic lipids complex the DNA and displace the intercalated
EtBr. When EtBr displacement is complete, the fluorescence reaches a
baseline. The isoelectric point, where the positive charges on the lipids
match the negative charges on the DNA, was determined as the onset
of the baseline value as described [32]. Fig. 2 displays the results of the
EtBr displacement assay for the CMVLs, plotting relative fluorescence
against the weight ratio of CMVL to DNA. Dashed vertical lines mark
the isoelectric point for each lipid. The resulting headgroup charges
are Z=1.4 for CMVL2, Z=1.9 for CMVL3, Z=2.4 for CMVL4, and
Z=3.1 for CMVL5, with an estimated accuracy of 0.1.
complexes exhibit a series of sharp peaks that are labeled q001, q002
,
q003, andq004. These are indicative of the lamellar structure, LαC, with a
repeat distance d=2π/q001. The peaks at qDNA (marked by arrows)
reflect the one-dimensional ordering of the DNA molecules within the
lamellar complex and correspond to an interhelical spacing
dDNA =2π/qDNA. The experimental values obtained in non-reducing
media are d=71.7 Å and dDNA =30.3 Å for CMVL2, and d=74.8 Å and
dDNA =27.3 Å for CMVL5. Complexes containing MVL5 (the non-
degradable analog of CMVL5) are also lamellar and their structure is
unaffected by the presence of a strong reducing agent such as DTT.
This is demonstrated in Fig. 5b, which shows the SAXS pattern
obtained for MVL5/DOPC–DNA complexes at ΦMVL5 =0.6 after
addition of DTT. The characteristic pattern of LαC complexes is
preserved, with d=71.3 Å and dDNA =27.3 Å. In contrast, addition
of reducing agents drastically changes the structure of CMVL/DOPC–
DNA complexes. As evident from the scans shown at the bottom of
Fig. 5, the characteristic peaks of the lamellar structure disappear after
treatment with DTT or GSH. In the case of CMVL2, only a single feature
remains: a broad peak at q≈0.143 Å−1 for DTT-treated complexes,
and a less symmetric, broad feature at about the same position for
GSH-treated complexes. In the case of CMVL5, similar broad features
are seen, albeit at a lower q≈0.135 Å−1. In addition, a sharp peak at
q=0.231 Å−1 appears for complexes treated with DTT, while a
broader peak at higher q is observed after treatment with GSH.
As confirmed by TLC analysis (see Supplementary data), reducing
agents such as GSH and DTT are able to reduce (break) the disulfide
spacer between the positively charged headgroup and the hydropho-
bic tail of the CMVLs. The SAXS data shows that the CMVLs are also
reduced when assembled into CL–DNA complexes, as indicated by the
disappearance of the lamellar structure (which remains intact under
the same conditions for non-degradable MVL5). The products of the
reductive cleavage of CMVL/DOPC–DNA complexes are DNA, multi-
valent ions (the former lipid headgroup), the lipid tail and DOPC.
Theoretical as well as experimental studies have shown that
multivalent ions are able to condense DNA into bundled aggregates
3.3. Characterization of CL–DNA complexes by dynamic light scattering
and optical microscopy
To unambiguously demonstrate that the lipids and DNA form
complexes and to further characterize these complexes, we per-
formed optical microscopy and dynamic light scattering (DLS)
experiments. Fig. 3 shows optical micrographs of CMVL5/DOPC–
DNA complexes with ΦDOPC =0.6 and L/D=10 (the optimal compo-
sition for transfection, see below). The differential interference
contrast image (showing all observable particles) and the two
fluorescence mode images (showing lipid and DNA, respectively)
fully overlap. This is demonstrated more quantitatively by the
intensity profiles obtained from the fluorescence mode images.
Thus, optical microscopy proves that all observed particles contain
both lipid and DNA.
Fig. 4 shows the results of DLS measurements of DNA complexes of
the CMVLs at the L/D optimal for transfection (see below),
corresponding to L/D=10.0 for CMVL5, L/D=10.7 for CMVL4,
L/D=20.2 for CMVL3, and L/D=13.7 for CMVL2. For all complexes,
the size increases with the mole fraction of CMVL. The measured
hydrodynamic diameter is between 100 and 200 nm for most of the
samples, and only high mole fractions of CMVL2 yield a diameter
larger than 300 nm. The size of complexes at a given mole fraction of
CMVL shrinks with headgroup valency for CMVL5 to CMVL3, but
CMVL2 deviates from this trend, forming the largest complexes by far,
in particular at high mole fractions of CMVL2.

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R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
OH
O
a)
b)
Cl-
Cl-
NH+3
OH
H3N+
S
S
S
O
1
HO
HO
OEt
c)
O
H2N
S
Boc
O
O
+
N
H
OH
3
DOB (2)
d)
e)
H
Boc
N
Boc
N
O
H
f)
S
NH+3
O
Boc
+
N
S
N
TFA-
H
HO
N
O
Boc
O
4
5
d)
e)
NH+3
TFA-
NH+3
O
H
N
NH+2
O
O
S
NH+2
N
H
S
O
CMVL4
Fig. 1. Synthesis of reductively degradable CMVL4, starting from oleic acid. Reaction conditions: (a) LiAlH4; (b) 1. PPh3, DEAD 2. KOH; (c) Boc2O, Et3N; (d) TBTU, DIEA; (e) TFA; (f) see
ref [28]. DEAD: diethylazodicarboxylate; DIEA: N,N-diisopropylethylamine; TBTU: O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TFA: trifluoroacetic acid.
if their valency, Z, is at least 3 [60–64]. Taken together with the EtBr
assay data, this means that only the cleaved headgroup of CMVL5 is
able to condense DNA. This permits assignment of the additional peak
observed after reductive treatment. The position of this peak is
consistent with the formation of hexagonal DNA bundles, as seen
with, e.g., spermine, spermidine, and small poly(propyleneimine)
dendrimers [65]. For the DTT-treated CMVL5/DOPC–DNA complexes,
the observed peak corresponds to an interhelical distance of
dDNA =4π/(3)1/2q=31.2 Å in the bundles. A detailed, quantitative
SAXS study of all CMVLs and their DNA complexes is in progress, and
will be reported in due course.
3.5. Cytotoxicity
Cytotoxicity was measured by assessing cell viability with a
tetrazolium salt-based assay [66]. Fig. 6a shows the resulting data for
DNA complexes (at ΦDOPC =0) of CMVLn (n=2 to 5), MVL5, DOTAP,
and Lipofectamine 2000 as a function of lipid/DNA weight ratio (to
accommodate Lipofectamine 2000). Up to a cationic lipid/DNA weight
ratio (L/D) of 18, none of the reagents exhibits appreciable
cytotoxicity, with CMVL4 and CMVL5 affecting cell viability the
least. Beyond this value, however, toxicity increases strongly for
DOTAP and Lipofectamine 2000, and somewhat less strongly for
CMVL2 and MVL5. In contrast, cell viability stays near 90% up to
L/D=28 for CMVL3 and L/D=36 for CMVL4 and CMVL5. Thus, with
the exception of CMVL2, the CMVLs showed a remarkable decrease in
cytotoxicity over important commercially available reagents as well
as MVL5, the CMVL5 analog without a reductively cleavable disulfide
bond.
Comparing optimal or near-optimal (for CMVL2 and CMVL3)
formulations, Fig. 6b shows cytotoxicity data taken at ΦDOPC =0.4 for
the CMVLs and MVL5 in comparison with the data for DOTAP and
Lipofectamine 2000 from Fig. 6a. The data is plotted as a function of
total lipid/DNA ratio (TL/D; for DOTAP and Lipofectamine 2000,
L/D=TL/D). The addition of neutral lipid extends the onset of toxicity
as a function of TL/D. This effect is strongest for CMVL2, which behaves
very similarly to the other CMVLs at this ΦDOPC. For MVL5, toxicity
increases sharply for TL/DN33; for the CMVLs, toxicity increases less
Fig. 2. Data from the EtBr displacement assay used to measure the charge of CMVLs in
CL–DNA complexes, obtained at ΦCMVL =1. The dashed lines indicate the location of the
isoelectric point, which was determined as described previously [32]. The resulting
headgroup charges are Z=1.4 for CMVL2, Z=1.9 for CMVL3, Z=2.4 for CMVL4, and
Z=3.1 for CMVL5, with an estimated accuracy of 0.1. Error bars show the standard
deviation of duplicate measurements.

R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
2163
Fig. 3. Optical micrographs of CMVL5/DOPC–DNA complexes at ΦCMVL5 =0.6 and L/D=10. (a) Differential interference contrast image. (b,c) Fluorescence mode images showing
lipid and DNA label, respectively. Scale bar, 10 μm (valid for all three micrographs). (d, e) Intensity profiles along the dotted lines in the fluorescence mode images of parts b and c.
Colocalization of the lipid and DNA fluorescence in all observed particles unambiguously demonstrates complex formation.
sharply and at much higher TL/D. At very high TL/D values, the toxicity
of the CMVLn lipids decreases with increasing n.
The comparison of MVL5 with CMVL5 is the most relevant in terms
a
b
of deducing structure–toxicity relationships, because the only
difference between those lipids is in the spacer, which in the case of
CMVL5 provides an established means of cleavage in the cytoplasm. In
addition, the fact that both CMVL3 and CMVL4 show a toxicity profile
similar to that of CMVL5 for both ΦDOPC investigated suggests that for
these three lipids, the headgroup size and charge play only a minor
role in determining the toxicity. On the other hand, CMVL2–DNA
complexes (at ΦDOPC =0) are unexpectedly toxic. The source of this
high toxicity is not clear, and a subject of ongoing investigations. We
q001
q001
q002
qDNA
q002
qDNA
q003
q004
q003
q004
qDNA-Bundle
Fig. 4. Hydrodynamic diameter (z-average) of CMVL/DOPC–DNA complexes at
Φ
CMVL =0.6 in Opti-MEM 20 min after complex formation. The samples were prepared
Fig. 5. SAXS data of CL–DNA complexes before and after addition of reducing agents.
(a) Data for CMVL2/DOPC–DNA complexes at ΦCMVL2 =0.6 as prepared (blue). (b) Data
for CMVL5/DOPC–DNA complexes at ΦCMVL5 =0.6 as prepared (blue) and after addition
of DTT (red) or GSH (green). Also shown is data for MVL5/DOPC–DNA complexes at
at values of L/D optimal for transfection (L/D=10.0 for CMVL5, L/D=10.7 for CMVL4,
L/D=20.2 for CMVL3, and L/D=13.7 for CMVL2, see below). Data points shown are the
average of duplicate measurements of the same sample, with error bars showing the
standard deviation.
ΦCMVL5 =0.6 after addition of DTT (brown).

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R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
TE for the MVLn lipid series (n=2, 3, 5) [32]. Lipofectamine 2000 was
used as per manufacturer's instructions and its TE as well as that of
optimized DOTAP/DOPC complexes is plotted for comparison. As
observed with other lipids [26,28,52,67], the TE of complexes of the
CMVLs and MVL5 initially increases with L/D, then reaches a plateau,
and (in the case of CMVL2 and CMVL5) finally drops again for even
higher L/D. The plateau of optimal TE spans L/D≈9–20 for CMVL2,
L/D≈18–24 for CMVL3, L/D≈10–21 for CMVL4, L/D≈7–13 for
CMVL5, and L/D=7–15 for MVL5. The lipids with higher valency
(CMVL4, CMVL5, MVL5) reach a maximum TE that is very high and
comparable to that of Lipofectamine 2000. The optimal TE for CMVL2
is only slightly lower than that for CMVL3, which in turn is about an
order of magnitude lower than that of the high-valency CMVLs.
To investigate the effect of membrane charge density, we varied
ΦDOPC at an optimal L/D. We chose L/D=10.0 for CMVL5, L/D=10.7
for CMVL4, L/D=13.7 for CMVL2, and L/D=7.4 for MVL5 (each
approximately corresponding to a lipid/DNA charge ratio of 6) as well
as L/D=20.2 for CMVL3 and L/D=5.9 for DOTAP. The results for TE at
the optimal L/D as a function of mole fraction of cationic lipid are
shown in Fig. 7b. As observed previously for DOTAP and the MVL
series [32], TE initially increases with increasing mole fraction of
cationic lipid to a maximum. For DOTAP, this maximum is at
ΦDOTAP =0.8 to 1. For the multivalent lipids, the maximum TE is
reached at a lower Φ(C)MVL of 0.6 for CMVL4 and CMVL5 and of 0.8 for
CMVL2 and CMVL3; TE then drops again. At the optimal composition,
TE for complexes prepared from CMVL2 and CMVL3 is slightly closer
to the TE achievable with CMVL4 and CMVL5 and exceeds the TE for
optimized DOTAP complexes. Thus, as observed for the MVL series
[32], the TE of CMVL/DOPC–DNA complexes exhibits three regimes as
a function of increasing ΦCMVL: initial increase, optimum TE, and a
decrease at very high σM. Regarding the transfection mechanism, this
suggests that lipid degradability (and, judging from the SAXS and
cytotoxicity results, most likely intracellular degradation) does not
affect the transfection mechanism. It appears that release of DNA from
the lipids is not the factor limiting TE, since the drop in TE is not only
observed for CMVL5 (for which condensation by the cleaved head-
group would still limit DNA release) but also for the other, less highly
charged CMVLs.
Fig. 6. (a) Cytotoxicity (in mouse L-cells) of CMVL–DNA complexes and other vectors at
Φ
DOPC =0. Cell viability is plotted as a function of lipid/DNA weight ratio (L/D).
(b) Cytotoxicity of CMVL–DNA complexes and other vectors for optimal or near-
optimal (for CMVL2 and CMVL3) formulation (ΦDOPC =0.4 for MVL5 and the CMVLs;
Φ
DOPC =0 for DOTAP and Lipofectamine 2000). Cell viability is plotted as a function of
total lipid/DNA weight ratio (TL/D; for DOTAP and Lipofectamine 2000, L/D=TL/D).
Note the change in scale for the x-axis. For both plots, error bars show the standard
deviation of quadruplicate measurements.
In the light of their reduced cytotoxicity, the finding that the
CMVLs with higher valency transfect cells as efficiently as non-
degradable MVL5 and benchmark commercial lipids represents a
major success. It suggests that the toxicity of a given lipid can be
effectively reduced without negative impact on its efficiency by
inserting a disulfide spacer between the lipid tails and headgroup.
have found that similar divalent lipids (MVL2 and a lipid with a
slightly different, longer spacer) are also unexpectedly toxic (MVL2 in
siRNA delivery) or unexpectedly inefficient DNA vectors. Thus, we
believe that the deviating behavior of CMVL2 is not related to an effect
of the spacer and rather due to effects specific to divalent (or possibly
ornithine-based) lipids. Of note, CMVL2 forms comparatively large
DNA complexes (cf. DLS results in Fig. 4), in particular at ΦDOPC =0
where it is most toxic. Summarizing the cytotoxicity results, the
design of the CMVLs, with the disulfide-spacer that connects head-
group and lipid tails acting as a built-in weak link that can be
reductively cleaved in the cytoplasm, appears to be successful in
reducing lipid and CL–DNA complex cytotoxicity. Of note, MVL5 has
previously been found to be a much better vector for gene silencing
using siRNA than DOTAP, in part due to the lower toxicity of MVL5 at
increased lipid/DNA charge ratios. The cleavable disulfide linker of
CMVL5 reduced this toxicity even further. Studies of the CMVLs as
siRNA vectors are currently ongoing.
4. Conclusions
We have presented the efficient synthesis of a series of new
degradable multivalent cationic lipids, termed CMVLn (n=2 to 5),
which contain a disulfide-bond spacer between headgroup and the
hydrophobic moiety. This spacer is cleaved by reducing agents such as
the biologically relevant reducing peptide glutathione or DTT. SAXS
demonstrates that the initially formed lamellar phase (LαC) of CMVLn–
DNA complexes completely disappears upon reductive cleavage of the
CMVLs. Importantly, while the CMVLs (n=3 to 5) transfect
mammalian cells with efficiencies comparable to highly efficient
non-degradable analogs as well as benchmark commercial reagents
such as Lipofectamine 2000, they do so at significantly reduced
cytotoxicity. DNA condensation by the cleaved head group—observed
for CMVL5, but not CMVL2—does not reduce the transfection
efficiency. This demonstrates that triggered disintegration of degrad-
able disulfide spacers can be used to reduce the cytotoxicity of
synthetic nonviral vectors without compromising their transfection
efficiency. The fact that transfection efficiency remains high also
shows that the key steps of lipid-mediated DNA delivery prior to
cytoplasmic release—DNA compaction, complex uptake and
3.6. Cell transfection
Transfection efficiency of CL–DNA complexes at varied L/D and
ΦDOPC was measured using a luciferase assay. Fig. 7 shows the
resulting data for complexes of CMVLn (n=2 to 5) and MVL5 at
Φ
Φ
DOPC =0.4 as a function of L/D. For the multivalent lipids,
DOPC =0.4 was chosen because it yielded optimal or near-optimal

R.S. Shirazi et al. / Biochimica et Biophysica Acta 1808 (2011) 2156–2166
2165
Fig. 7. Transfection efficiency of CMVL-based CL–DNA complexes compared with the non-degradable lipid MVL5 and commercial vectors. The amount of DNA was constant for all
data points. (Top) TE as a function of L/D at a constant ΦDOPC =0.4. TE for DOTAP and Lipofectamine 2000 is for optimized formulations. (Bottom) TE at optimal L/D as a function of
mole fraction of cationic lipid. Specifically, data was taken at L/D=10.0 for CMVL5, L/D=10.7 for CMVL4, L/D=13.7 for CMVL2, and L/D=7.4 for MVL5 (each approximately
corresponding to a lipid/DNA charge ratio of 6) as well as L/D=20.2 for CMVL3 and L/D=5.9 for DOTAP. For both plots, error bars show the standard deviation of duplicate
measurements.
endosomal escape [32,33]—are not compromised by the lipid degrada-
Appendix A. Supplementary data
tion process.
Supplementary data to this article can be found online at doi:10.
1016/j.bbamem.2011.04.020.
Acknowledgements
This work was supported by NIH GM-59288. Cecília Leal was
funded by the Swedish Research Council (VR) and in part by DOE
BESDE-FG-02-06ER46314. Some aspects of the synthesis involving
design of environmentally responsive lipid materials were also
supported by BESDE-FG-02-06ER46314. NMR characterization was
performed using the Central Facilities of the Materials Research
Laboratory at UCSB which are supported by the MRSEC Program of the
NSF under award no. DMR05-20415; a member of the NSF-funded
Materials Research Facilities Network (www.mrfn.org). SAXS exper-
iments were carried out at the Stanford Synchrotron Radiation
Lightsource, which is supported by the Department of Energy.
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