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Y. Sun et al. / International Journal of Pharmaceutics 468 (2014) 83–90
chemicals, including cationic lipids (Martin et al., 2005), cationic
polymers (Boussif et al.,1995; Furgeson et al., 2003; Suh et al.,1994;
Xu and Yang, 2011), cell-penetrating peptides (CPPs) (Furgeson
et al., 2003; Parente et al.,1990; Suh et al.,1994; Trabulo et al., 2010)
and some dendrimers (Furgeson et al., 2003; Kukowska-Latallo
et al.,1996; Suh et al.,1994; Zhou et al., 2006) havebecome the most
widely used vectors in biological studies and pre-clinical gene
therapies.
showed best pDNA transfection efficiency which is comparable to
the commercial gene delivery vector PEI-25k, but its cytotoxicity
was much lower. With acceptable gene transfection efficiency and
low cytotoxicity, this cationic amphiphilic lipopeptide could be
potentially applied in gene therapy.
2. Materials and methods
The basic principle of non-viral vectors mediated transfection is
as follows: (i) Non-viral vectors compact large DNA molecules into
small particles by electrostatic interactions between the positively
charged vectors and negatively charged DNA molecules (Furgeson
et al., 2003; Gershon et al., 1993; Ma et al., 2007; Suh et al., 1994).
The formation of vector/DNA complex can also protect DNA from
degradation by enzymes in the external environment. (ii) An excess
addition of cationic vectors equip the complex surfaces with
positive charges, which is presumed to facilitate subsequent
cellular uptake via interaction with the negative cell surface
structures such as heparin sulphates and other proteoglycans
2.1. Materials
Dodecanol, tetradecanol, hexadecanol and cholesteryl chlor-
oformate were purchased from Alfa Aesar Co. (Ward Hill, MA).
Fmoc-Lys(Boc)-Wang resin, H-His(Trt)-2-Chlorotrityl resin,
0
0
Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, O-benzotriazole-N,N,N ,N
-tetra-methyluroniumhexafluorophosphate (HBTU), N-hydroxy-
benzotriazole (HOBt), piperdine, diisopropylethylamine (DIEA),
thioanisole, dithioglycol and trifluoroacetic acid (TFA) were
obtained from GL Biochem (Shanghai) Ltd. (Shanghai, China).
Dimethylformamide (DMF) and dichloromethane (DCM) were
provided by DiKMA Technologies Inc. (Beijing, China). Branched
poly(ethylene imine) (PEI-25k, Mw = 25,000) was supplied by
Sigma–Aldrich Co. (St. Louis, MO). Dulbecco’s Modified Eagle’s
Medium (DMEM), fetal bovine serum (FBS), penicillin–streptomy-
cin were purchased from Gibco (Grand Island, NY). Dimethylsulf-
oxide (DMSO) and thiazoyl blue tetrazolium bromide (MTT) were
purchased from BioDee (Beijing, China). pEGFP-N2 and 293T cell
line were generously provided by the laboratory of Prof. Yanmei Li
(School of Life Science, Tsinghua University).
(Mislick and Baldeschwieler, 1996). These complexes are taken up
by cells through clathrin-mediated endocytosis, caveolae-mediated
endocytosis and macropinocytosis (Conner and Schmid, 2003;
Kirkham and Parton, 2005; Nichols, 2003; Suh et al., 1994). (iii)
Once in the endocytic pathway, the plasmids may be degraded
when reaching the lysosomes. Accordingly, for effective trans-
fections, the plasmids need to acquire cytosolic access at an earlier
stage by escaping from the endosomes (Mukherjee et al., 1997).
Different mechanisms such as pore formation in the endosomal
membranes (Huang et al., 2004), pH-buffering effect of protonable
groups (Moreira et al., 2009; Pack et al., 2000; Suh et al., 1994;
Varkouhi et al., 2011) and fusion into the lipid bilayer of endosomes
2.2. Synthesis of the lipopeptides
(
Xu and Szoka, 1996) have been proposed to facilitate the
The N-phosphoryl oligopeptides were synthesized from phos-
phites and oligopeptides, which could be divided into two major
steps: synthesis of phosphites and its incorporation to the
oligopeptides (Ma and Zhao, 1992; Moreira et al., 2009; Musiol
et al.,1994; Suh et al.,1994). The synthesis of phosphites with long
dialkoxyl chains was obtained through direct esterification of
phosphorus trichloride by 3 equivalents of alcohol in benzene
(Fig. 1). Separation of the products was accomplished by reduced
pressure distillation.
Peptides were synthesized on resin utilizing standard Fmoc
solid phase procedure. Phosphites (Fig. 1A) and cholesteryl
chloroformate (Fig. 1B) were incorporated as the last amino acid.
The resulting dry resin bound lipopeptides were cleaved and side-
endosomal escape.
Felgner et al. (Felgner et al., 1987) reported the first cationic
lipid named DOTMA (N-(1-(2,3-dioleyloxy) propyl)-N,N,N-tri-
methyl-ammonium chloride), which consists of a quaternary
amine connected to two unsaturated aliphatic hydrocarbon chains
via ether groups. DOTMA was 5 to over 100 folds more effective
than either the DEAE-dextran or the calcium phosphate transfec-
tion technique. Cationic lipids are amphiphilic molecules that
consist of the following three structural segments: (i) a hydrophilic
headgroup which is positively charged, usually via the protonation
of monovalent or multivalent amine groups; (ii) a hydrophobic
region composed of a steroid or alkyl chains (saturated or
unsaturated); (iii) a linker connecting the cationic headgroup
with the hydrophobic anchor, whose nature and length may
impact on the stability and biodegradability of the vector.
Cell-penetrating peptides (CPPs) are short peptides with
potential abilities to translocate across the plasma membrane of
living cells. Many CPPs are derived from the transduction domains
of viral proteins involved in interaction with cell membranes (Vivès
et al., 1997). A typical example of CPPs is HIV Tat (Ignatovich et al.,
chains were deprotected using a cocktail of TFA:H
phenol:ethanedithiol (82.5:5:5:5:2.5). The resin was then removed
by filtration, and TFA was evaporated with a slow stream of N . The
2
O:thioanisole:
2
lipopeptides were precipitated from the filtrate with cold diethyl
ether. The resulting crude lipopeptides were then purified by RP-
HPLC (Cosmosil C18 & C4 peptide/protein column). Purified
lipopeptide solutions were evaporated to remove organic phase
and then lyophilized, obtaining a pure lipopeptide powder utilized
for all subsequent experiments. The purity (>90%) of each
lipopeptide was assessed by analytical HPLC and ESI-MS.
2
003) consisting of 6 arginines and 2 lysines in its 13 amino acid
residues, which is effective in binding to plasmid DNA through
charge interaction and condensing it. CPPs have been studied
during the last decades as materials for drug delivery vehicles due
to their biodegradability, biocompatibility, low toxicity and ease of
synthesis (Torchilin et al., 2003).
In this study, a series of cationic amphiphilic N-phosphoryl
oligopeptides (C12-K6, C14-K6, C16-K6, Chol-K6 and C12-H6) were
designed and synthesized using the phosphite method (Ma and
Zhao, 1992; Moreira et al., 2009; Musiol et al., 1994; Suh et al.,
2.3. Agarose gel retardation assay
To examine the ability of each cationic lipopeptide to bind
pDNA, an agarose gel retardation assay was performed (Weyland
et al., 2013). The complex solution was prepared under a
predetermined N/P charge ratio (the molar ratio of the amine
groups in the lipopeptides to the phosphates in pDNA) by mixing
1994) and Fmoc solid-phase synthesis. Results showed that the
pDNA (0.8 mg pDNA in 8 ml TE buffer) with an equal volume of the
length and structures of the N-terminal phosphoryl ester
chains could dramatically affect the transfection efficiency of
lipopeptide/pDNA complexes. At an optimal P/N ratio of 20, C12-K6
lipopeptides in PBS buffer solution (pH 7.4). Each mixture was
vortexed for 30 s and incubated for 30 min at room temperature.
Then, the mixture was analyzed on a 0.8% agarose gel containing