Tri-peptide cationic lipids for gene delivery

Article abstract of DOI:10.1039/c4tb01312c

Several novel tri-peptide cationic lipids were designed and synthesized for delivering DNA and siRNA. They have tri-lysine and tri-ornithine as headgroups, a carbamate group as a linker and 12 and 14 carbon atom alkyl groups as tails. These tri-peptide cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. Their particle size, zeta potential and DNA-binding were characterized to show that they were suitable for gene transfection. Further results indicate that these lipids can transfer DNA and siRNA very efficiently into NCI-H460 and Hep-2 tumor cells. The selected lipid, CDO14, was able to deliver combined siRNAs against c-Myc and VEGF for silencing distinct oncogenic pathways in lung tumors of mice, with little in vitro and in vivo toxicity. This journal is

Full text of DOI:10.1039/c4tb01312c

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Tri-peptide cationic lipids for gene delivery†
Yinan Zhao,ab Shubiao Zhang, Yuan Zhang,c Shaohui Cui,b Huiying Chen,b
b
*
Cite this: J. Mater. Chem. B, 2015, 3,
119
b
d
a
Defu Zhi, Yuhong Zhen, Shufen Zhang and Leaf Huange
*
Several novel tri-peptide cationic lipids were designed and synthesized for delivering DNA and siRNA. They
have tri-lysine and tri-ornithine as headgroups, a carbamate group as a linker and 12 and 14 carbon atom
alkyl groups as tails. These tri-peptide cationic lipids were prepared into cationic liposomes for the study of
the physicochemical properties and gene delivery. Their particle size, zeta potential and DNA-binding were
characterized to show that they were suitable for gene transfection. Further results indicate that these lipids
can transfer DNA and siRNA very efficiently into NCI-H460 and Hep-2 tumor cells. The selected lipid,
CDO14, was able to deliver combined siRNAs against c-Myc and VEGF for silencing distinct oncogenic
pathways in lung tumors of mice, with little in vitro and in vivo toxicity.
Received 7th August 2014
Accepted 24th September 2014
DOI: 10.1039/c4tb01312c
www.rsc.org/MaterialsB
found that the introduction of peptide groups into lipids can
prolong their half-life in vivo and enhance targeting thereof.21
1 Introduction
Therefore, peptide based cationic lipids have shown more
superiorities over other cationic lipids, such as good biode-
gradability, excellent biocompatibility and targeting ability to
cells, and potential applications in improving the delivery of
gene therapeutics.22–24 However, the current peptide based
cationic lipids still hold some limitations.25,26 Therefore, we
designed and synthesized a series of novel tri-peptide cationic
lipids CDL12, CDL14, CDO12 and CDO14 (Fig. 1) for facilitating
the use of peptide based lipids for gene delivery.
Viral vectors and non-viral vectors are widely used to modulate
gene expression in target tissues to correct genetic defects for
gene therapeutic applications. Viral vectors, such as adenovirus
vectors, adeno-associated virus vectors, or retrovirus vectors,
have been applied in clinical trials.1–3 However, some side
effects including immunoresponse and infection have been
reported.4,5 Non-viral gene delivery systems have, on one hand,
been demonstrated as standard tools for in vitro transfection
using many commercially available kits.6–8 On the other hand,
they are regarded as a promising approach for the treatment of
genetic diseases and cancers.9–12
Among these non-viral vectors, peptide cationic lipids have
drawn great attention,13–18 as peptides are more biocompatible.
Peptides and their derivatives have been used for restraining
cancer cell migration, curing anti-thrombosis, treating acute renal
failure, reducing anti-inammation, promoting skin regenera-
tion, etc. But the isolated small peptides are easily degraded by
enzymes in vivo, and they could be cleared very quickly.19,20 People
2 Experimental procedures
Materials
9-Fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu),
o-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexa-
uorophosphate (HATU), lauryl alcohol, myristyl alcohol,
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian
116021, Liaoning, China. E-mail: zhangshf@dlut.edu.cn; Tel: +86-411-84986265
bSEAC-ME Key Laboratory of Biotechnology and Bio-resources Utilization, Dalian
Nationalities University, Dalian 116600, Liaoning, China. E-mail: zsb@dlnu.edu.cn;
Fax: +86-411-87656141; Tel: +86-411-87656141
cDepartment of Materials Science and Engineering, Department of Biological
Engineering, The David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
dCollege of Pharmacy, Dalian Medical University, Dalian 116044, Liaoning, China
eDivision of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of
North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
† Electronic supplementary information (ESI) available: IR, NMR, HR-ESI-MS and
HPLC characterisation of all the lipids synthesized; TEM images of the CDO14
liposome and CDO14 liposome/DNA lipoplex; gel electrophoresis of
liposome/pDNA complexes; in vitro transfection images against NCI-H460 and
Hep-2 cells. See DOI: 10.1039/c4tb01312c
Fig. 1 Structures of tri-peptide cationic lipids CDL12, CDL14, CDO12
and CDO14.
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N,N0-carbonyldiimidazole (CDI), diethylenetriamine, 3-amino-
1,2-propanediol, lysine and ornithine were purchased from
Shanghai Aladdin Industrial Inc. (China). Lipofectamine 2000
was purchased from Invitrogen Life Technologies (USA). 1,2-
Dioleoyl-3-trimethylammonium-propane chloride salt (DOPE)
and anti-luciferase siRNA were purchased from Sigma-Aldrich
(USA). 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP)
was purchased from Roche Shanghai (China). Hep-2 and NCI-
H460 cells were purchased from the Institute of Biochemistry
and Cell Biology (China). A549 cells were from Leaf's lab of the
University of North Carolina at Chapel Hill. DMEM, RPMI1640,
fetal bovine serum (FBS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from
Gibco (USA). Green uorescent protein (pGFP-N2) and lucif-
erase (pGL3) plasmid vectors were purchased from Sangon
Biotech Co. Ltd. (China), and extracted in our lab. The Bright-
Glo™ Luciferase assay system was purchased from Promega
Biotech Co. Ltd. (Beijing, China). All other chemicals were of
reagent grade.
Synthesis of lipids
The synthesis of these lipids was performed as follows, using
CDL12 as an example (Scheme 1): CDI was added to lauryl
alcohol in toluene and kept at reux for 3 h. Diethylenetriamine
was added. Aer another 3 h, the solvent was evaporated. The
crude product was recrystallised from the mixture solvent of
ethanol–water to give intermediate 1. Boc2O in acetone was
added to L-Lys aqueous solution. CuSO4$5H2O was added and
kept stirring for 14 h at room temperature. Anhydrous sodium
carbonate, 8-hydroxyquinoline and Fmoc-OSu were added. The
solution was stirred at room temperature for 6–7 h before being
ltered. The ltrate was cooled to 0 ꢀC, and pH was adjusted to
2–3 with HCl solution. The crude product was recrystallised
from the mixture solvent of acetonitrile and petroleum ether to
give intermediate 2 as Fmoc-Lys (Boc)-OH.
For the construction of CDL12, intermediate 2 was added to
HATU in dichloromethane. Aer a reaction for 1 h, a dichloro-
methane solution of intermediate 1 was added. The Boc group
was removed with CHCl3/TFA (1 : 1) and the Fmoc group with
dioxane/NaHCO3 to give intermediate 3 as H-Lys-OCD12. The
reaction was repeated as the above steps to give the crude
product with intermediates 2 and 3. The resulting sticky paste
was recrystallised from the mixed solvent of acetonitrile and
petroleum ether to give a white crystal as CDL12.
Scheme 1 Synthesis of tri-peptide lipid CDL12. Reaction conditions:
(A) CH2Cl2, 40 ꢀC, 3 h; (B) THF, 40 ꢀC, 3 h; (C) H2O, 27 ꢀC, 30 h; (D)
room temperature, 4 h; (E), (H), and (I) CH2Cl2, 20 ꢀC, 12 h; (F) and (J)
CHCl3, 4 ꢀC, 2 h; (G) and (K) dioxane, room temperature, 2 h. Reagents:
(Boc)2O ¼ di-tert-butyl dicarbonate, Fmoc-OSu ¼ 9-fluorenylmethyl-
succinimidyl-carbonate, HATU
¼
azabenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate, TFA ¼ trifluoroacetic acid.
Preparation and characterisation of liposomes
The protocol for preparation of liposomes based on CDL12,
CDL14, CDO12 and CDO14 lipids was optimised to obtain high-
quality delivery systems. The lipids were formulated in combi-
nation with the neutral lipid DOPE in a 1 : 1 molar ratio. To
prepare liposomes, suitable amounts of lipid and DOPE were
dissolved in 1 mL of chloroform in a 5 mL glass vial. The solvent
was removed under a stream of nitrogen gas, followed by high-
vacuum desiccation. The dry lipid lm was resuspended in 1 mL
distilled water to give liposomes in a concentration of approx-
imately 1 mg mLꢁ1. The liposome/DNA complexes of varying
weight ratios (N/P) were prepared by mixing liposomes with
plasmid solutions 20 min before use.
Chemical analysis of lipids
1H and 13C NMR spectra were recorded at 400 and 100 MHz,
respectively, on a Varian MercuryPlus 400 spectrometer. High
resolution electrospray ionization mass spectrometry was per-
formed using a Q-Tof Micro (Micromass Inc., Manchester, UK).
IR spectra were recorded using an IRPrestige-21 (Shimadzu,
Japan). HPLC was performed on a LC-20AB (Shimadzu, Japan)
with a C18 column, 1% TFA in the methanol–water mobile
phase (85 : 15), at a ow rate of 0.8 mL minꢁ1
.
120 | J. Mater. Chem. B, 2015, 3, 119–126
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5% CO2 for 24 h to get a conuence of about 80%. Aer the
replacement of the medium, the liposome/DNA complexes were
added to the cells for further culture at 37 ꢀC under 5% CO2 for
24 h, and then MTT (20 mL, 5 mg mLꢁ1) was added and kept for
4.5 h. The absorbance at 570 nm was monitored by using the
enzyme mark instrument (Sunrise Tecan, Australia).
Particle size and zeta potential measurements
The particle size and zeta potential were measured with a
Malvern ZetaSizer Nano series (Westborough, MA) at room
temperature. All the lipoplexes of varying N/P ratios ranging
from 1 to 8 were prepared with pGL3 plasmid. For the
measurement of particle size and zeta potential, 20 mL of the
liposomes or lipoplexes were diluted to 1 mL with distilled
water.
Animals
In vivo studies were carried out on BALB/c nude mice 4–6 weeks
of age. All experiments performed on the animals were in
accordance with and approved by the Institutional Animal Care
and Use Committee at University of North Carolina.
Agarose gel retardation assay
The lipoplexes of varying N/P ratios ranging from 0 to 8 were
prepared with pGL3 plasmid. The lipoplexes were electro-
phoresed in 1.2% agarose gel containing GelRed and Tris-
acetate (TAE) running buffer at 90 V for 40 min. DNA bands were
visualised in a gel documentation unit (Syngene, Britain).
In vivo gene silencing study
Combined siRNAs against c-Myc and VEGF (1 : 1) were co-de-
livered by the CDO14 liposome. The tumor-bearing mice were
injected 4 times every 3 days via tail vein with lipoplexes at a N/P
ratio of 3 : 1 (with a dose of 0.45 mg total siRNA per kg). One day
aer the third injection, the mice were killed and the tumor-
loaded lungs were collected for the preparation of paraffin
embedded sections (4–5 mm thick). Expressions of c-Myc and
VEGF in the sections were examined immunohistochemically
using the antibodies from a kit in accordance with the product
protocol.
In vitro pDNA transfection
Hep-2 and NCI-H460 cells were seeded in 24-well plates (5.0 ꢂ 104
cells per well), and incubated at 37 ꢀC under 5% CO2 until
approximately 80% conuence was attained. The medium was
removed and replaced with 100 mL serum-free DMEM per well.
Cationic liposome/DNA lipoplexes were then added to the plates
ꢀ
and incubated for 4 h at 37 C. The medium was then replaced
with DMEM containing 10% FBS and 1% antibiotics, and the
cultures were maintained at 37 ꢀC under 5% CO2 for 48 h.
The expression of green uorescent protein was measured
using an inverted uorescence microscope (Olympus IX71,
Japan). Relative luciferase activity was assessed using a Bright-
Glo™ Luciferase Assay System and a Synergy™ 2 Multi-Detec-
tion Microplate Reader. Briey, the growth medium was
removed from each well, and luciferase activity was measured
aer lysis buffer was added. The total protein concentration in
the cell lysate was measured using a BCA protein assay kit and
luciferase activity was expressed as relative light units (RLU) per
mg protein.
TUNEL assay
TUNEL staining was performed as recommended by the man-
ufacturer's protocol (Promega, Madison, WI). Tumor-bearing
mice were given i.v. injections of the siRNA formulated in the
CDO14 on days 9 and 10. Twenty-four hours aer the second
injection, the mice were killed, and the tumor-loaded lungs
were collected for the TUNEL staining. The nuclei were coun-
terstained with hematoxylin, and the samples were imaged
using an inverted uorescence microscope (Olympus IX71,
Japan).
In vitro siRNA transfection
Toxicity of the cationic liposomes in vivo
A549 cells expressing rey luciferase were seeded in 12-well
plates (1.0 ꢂ 105 per well) approximately 24 h before experi-
ments. Cells were treated with different lipid formulations at an
anti-luciferase siRNA concentration of 40 nM in Opti-MEM at 37
ꢀC for 4 h. Cells were washed with DPBS, followed by incubation
with lysis buffer at room temperature for 20 min.
The tumor-bearing mice were i.v. injected with siRNA formu-
lated in the targeted liposome at 3 : 1 doses, and the serum
cytokine level was determined with an Olympus AU400 Auto-
matic Biochemistry Analyzer. The mouse body weight was also
monitored.
Fluorescence intensity in cell lysate was determined using a
Perkin-Elmer LS 50B luminescence spectrometer (Norwalk, CT)
(lex, 494 nm; lem, 519 nm). Cell lysate (5 mg per well) was dis-
solved in BCA protein assay reagent, and total protein concen-
trations were determined at 570 nm. The silencing rate is
expressed as relative light units (RLU) per microgram of total
protein.
Statistical analysis
The data were presented as mean values ꢃ SD. The statistical
signicance was determined using two-way analysis of variance.
P values <0.05 were considered signicant.
3 Results and discussion
Design and synthesis of cationic lipids
Cytotoxicity of the cationic liposomes
The tri-peptide cationic lipids designed are composed of three
The assay was performed in 96-well plates by maintaining the N/ biocompatible molecular moieties: tri-lysine peptides or tri-
P weight ratios as used in the transfection experiments. The ornithine peptides (the cationic headgroup), carbamate (the
cells were seeded in 96-well plates and incubated at 37 ꢀC under linker), and two hydrocarbon chains (hydrophobic tails). These
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tri-peptide cationic lipids were prepared by adapting
a
straightforward procedure (Scheme 1). Intermediate 1 was
obtained from carbonyldiimidazole, dodecyl alcohol and trie-
thylenetetraamine. The synthesis of intermediate 2 started with
Fmoc and Boc group protected lysine. Aer the condensation
under HATU activation and the deprotection by using TFA and
NaHCO3, intermediate 3 could be obtained. Following the same
procedure by using another 2 mol Boc and Fmoc protected
lysine; we got tri-peptide cationic lipids for further chemical
analysis and biological study.
The structures and purities of tri-peptide cationic lipids were
characterised by electrospray ionisation-mass spectrometry
(ESI-MS), proton nuclear magnetic resonance spectroscopy
(1H NMR, 13C NMR), infrared (IR) spectroscopy, and high
pressure liquid chromatography (HPLC) (Fig. S1–S4, ESI†). The
analytical data of these compounds are listed under the gures
in the ESI† to show that tri-peptide cationic lipids were
successfully obtained.
Particle size, and zeta-potential of tri-peptide cationic
liposomes and lipoplexes
Our previous study showed that liposomes had higher trans-
fection efficiency with an equivalent molar ratio of DOPE, so the
liposomes were prepared by using the tri-peptides with DOPE as
a co-lipid at a molar ratio of 1 : 1. The dynamic light scattering
showed that the particle sizes of these liposomes were from
70 nm to 150 nm, those of the lipoplexes of CDO14/DNA were
from 200 nm to 400 nm in the N/P ratio range of 1 : 1 to 8 : 1
(Fig. 2 and 3). Zeta potentials of liposomes were between 40 and
60 mV, which ensured the stability of the liposomes for at least
three months. Zeta potentials of the lipoplexes of CDO14/DNA
were found to increase from ꢁ50 mV to nearly 0 mV with the
increase of the N/P ratios from 1 : 1 to 8 : 1 (Fig. 2 and 3). The
sizes and zeta potentials were in the ranges suitable for gene
transfection.27 The morphological characteristics were directly
visualised by transmission electron microscopy (TEM) (Fig. S5,
Fig. 2 Particle size and zeta potential of tri-peptide liposomes. Lipo-
somes (20 mL) were diluted in 1 mL distilled water. Particle size (A) and
zeta potential (B) were measured using a Malvern ZetaSizer. The PDI of
CDL12, CDL14, CDO12, CDO14, lipofectamine 2000 and DOTAP was
0.286, 0.262, 0.296, 0.422, 0.306 and 0.242, respectively.
ensured the entry of pDNA into cells and the protection of pDNA
from degradation by DNase. (Fig. S6, ESI†).
In vitro pDNA and siRNA transfection
ESI†). Images obtained from the CDO14 liposome and CDO14 The in vitro transfection of these liposomes was evaluated using
liposome/DNA lipoplex at a N/P ratio of 3 : 1 revealed well- pGFP-N2 plasmid DNA and pGL3 plasmid DNA against Hep-2
dened spherical structures.
and NCI-H460 cells. The expression of pGFP-N2 and pGL3 is
illustrated in Fig. 4 (for more details see Fig. S7 and S8, ESI†)
and Fig. 5, respectively. The results indicate that all the lipids
were comparable to lipofectamine 2000 and DOTAP in terms of
DNA-binding assay
DNA-binding with liposomes is a key factor for gene trans- GFP and luciferase expression. They had higher transfection
fection.28 Therefore, the electrostatic binding interactions against Hep-2 cells than against NCI-H460 cells. The liposomes
between plasmid DNA and the tri-peptide cationic liposomes with the ornithine headgroup also showed higher transfection
were measured by the gel retardation assay across the N/P ratios than those with the lysine headgroup, perhaps because they
of 0 : 1–8 : 1. Findings in the gel retardation assay revealed all have smaller heads.
the liposomes could start to retard the plasmid DNA at a N/P
Through the above experiments, the transfection in cells will
ratio of 0.5 : 1. They could nearly completely retard pDNA at undergo the following process. First, the plasmid DNA binds to
higher N/P ratios of 6 : 1, 6 : 1, 6 : 1 and 3 : 1 for CDL12, CDL14, the tri-peptide head-groups of the cationic lipids. Then, the
CDO12 and CDO14, respectively. Though all the above lipo- lipid/DNA lipoplexes will enter the Hep-2 cells and NCI-H460
somes could bind pDNA at higher N/P ratios, the lower N/P ratio cells (e.g., via endocytosis), and trypsin in the cell will hydrolyze
of CDO14 for retardation compared with other liposomes may the carbamate bond in the lipids. The hydrolization assists DNA
reduce its dose required to obtain maximal transfection effi- to escape from the endosome for the subsequent transcription.
ciency. The above results demonstrate that cationic liposomes The whole process is readily performed and thus enhances DNA
can form DNA/liposomes complexes at different N/P ratios. This transfection efficiency.
122 | J. Mater. Chem. B, 2015, 3, 119–126
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