Design, synthesis and in vitro evaluation of d-glucose-based cationic glycolipids for gene delivery

Article abstract of DOI:10.1039/c5ob02107c

A cationic lipid consists of a hydrophilic headgroup, backbone and hydrophobic tails which have an immense influence on the transfection efficiency of the lipid. In this paper, two novel series of cationic cyclic glycolipids with a quaternary ammonium headgroup and different-length hydrophobic tails (dodecyl, tetradecyl, hexadecyl) have been designed and synthesized for gene delivery. One contains lipids 1-3 with two hydrophobic alkyl chains linked to the glucose ring directly via an ether link. The other contains lipids 4-6 with two hydrophobic chains on the positively charged nitrogen atoms. All of the lipids were characterized for their ability to bind to DNA, size, ζ-potential, and toxicity. Atomic force microscopy showed that the lipids and DNA-lipid complexes were sphere-like forms. The lipids were used to transfer enhanced green fluorescent protein (EGFP-C3) to HEK293 cells without a helper lipid, the results indicated that lipids 4-6 have better transfection efficiency, in particular lipids 5-6 have similar or better efficiency, compared with the commercial transfection reagent lipofectamine 2000.

Full text of DOI:10.1039/c5ob02107c

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DOI: 10.1039/C5OB02107C
Design, Synthesis and in vitro Evaluation of D-Glucose-Based Cationic Glycolipids for Gene
Delivery
Chengxi Hea, Shang Wangb, Meiyan Liua, Chunyan zhaoa, Shuanglin Xiangb, Youlin Zenga*
a
National & Local Joint Engineering Laboratory for New Petro-chemical Materials and Fine Utilization of
Resources, Hunan Normal University, Changsha, Hunan, 410081, P. R. China.
b
Key Laboratory of Protein Chemistry and Developmental Biology of State Education Ministry of China, College
of Life Sciences, Hunan Normal University, Changsha, Hunan, 410081, P. R. China.
Abstract
The cationic lipid consists of hydrophilic headgroup, backbone and hydrophobic tails which
have an immense influence on the transfection efficiency of the lipid. In this paper, two novel
series of cationic cyclic glycolipids with quaternary ammonium headgroup and different-length
hydrophobic tails (dodecyl, tetradecyl, hexadecyl) have been designed and synthesized for gene
delivery. One contains lipids 1-3 with two hydrophobic alkyl chains linked to glucose cycle
directly in ether. The other contains lipids 4-6 with two hydrophobic chains in the positively
charged nitrogen atoms. All of the lipids were characterized for their ability to bind to DNA, size,
ζ-potential, and toxicity. The atomic force microscope showed that the lipids and DNA-lipid
complexes were sphere-like forms. The lipids were used to transfer enhanced green fluorescent
protein (EGFP-C3) to HEK293 cells without helper lipid, the results indicated that lipids 4-6 have
better transfection efficiency, especially the lipids 5-6 have similar or better efficiency, compared
with commercial transfection reagent lipofectamine 2000.
1. Introduction
It has been proved that gene therapy has shown a prodigious potential to the treatment of a
[1-4]
variety of human diseases such as cancer, AIDS, viral infection, cardiovascular
and retinal
disease [5-7]. However, one of the most important requirements for gene therapy is to develop a
safe and efficient gene delivery system. Viral and non-viral vectors were developed as two major
types of carriers used in vitro and in vivo gene transfection. Viral vectors comprise retroviruses
[8-10], adenoviruses [11-12] and adeno-associated [13]. Viruses have shown high transfection efficiency
and have been applied to many clinical trials, but their safety has always been worried. In addition,
[14]
viral vectors have a low insert-size limit for the therapeutic genes they can pack inside
.
Consequently, increasing attention has been focused on non-viral vectors, including cationic lipids
[18-20]
[15], polymers [16-17], dendrimers
and nanoparticles [21]. However, unlike viral analogues, the
non-viral vectors have not evolved means to overcome cellular barriers and immune defense
mechanisms. The vitro cellular barriers for cellular uptake contain (a) adsorption of the lipid-DNA
complex to the cell surface [22]; (b) uptake of the complex, mainly by endocytosis into the cells
[26]
[23-24]; (c) release of DNA from the endosomes [25]; (d) the half-life of DNA in the cytoplasm
;
and (e) delivery of the DNA into the nucleus [27]. Because of the impedance of numerous
extracellular and intracellular obstacles, non-viral gene carriers consistently exhibit significantly
reduced transfection efficiency. However, biocompatibility and potentiality of large-scale
substances binding make these non-virus vectors increasingly attractive for gene therapy. Among
these arsenals of non-viral transfection vectors, cationic lipids hold promise for: (a) their
*
Corresponding author. Tel.:+86 13975805056; fax: +86 73188872531
E-mail address: youlinzengcn@gmail.com (Youlin Zeng).
1

Organic & Biomolecular Chemistry
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reproducibility and simplicity in preparation, (b) their non-immunogenic nature andDO(cI:)10t.h10e3i9r/C5OB02107C
efficiency in forming stable injectable complexes even with large DNA [28]
.
Since the first cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
(DOTMA) was synthetized and applied to gene transfection as novel non-viral reagents reported
by Felgner et al. in 1987 [29], a large number of cationic lipids have been synthesized and
investigated, including cholesterol-based lipids [30-31], amino acids-based lipids [32-34], glycerol
based lipids [35-36], tocopherol based lipids [37-38], and sugar based lipids [39-43]. In recent years, it
has become more and more active to develop novel cationic glycolipids as gene transfection
reagent and investigate their capacity of deliver nucleic acids into the cells. Previous studies have
focused mainly on the relationship between transfection efficiency and structure of cationic
glycolipids. Primarily, R. Banerjee, et al. prepared and evaluated open-cycle D-xylose and
D-arabinose-based glycolipids with quaternary ammonium cationic headgroup, and have found
that additional hydroxyl groups attached to the hydrophilic terminal of the cationic glycolipids can
improve the transfection efficiency and decrease the cell cytotoxicity [39]. Posteriorly, Mahidhar V.
Y. and coworkers discovered that the transfection efficiency of the open-form galactosylated
cationic glycolipids was strikingly dependent on the spacer-arm between the open-form galactose
and the positively charged nitrogen atom in the headgroup region [40]. Similarly, Mukthavaram R.,
et al. synthesized two series of cationic glycolipids with cyclic and open D-galactose heads
containing varying spacer-arm lengths between the sugar and positively charged nitrogen atoms,
and it was indicated that cationic glycolipids with cyclic sugar-head require longer spacer arms
than their acyclic sugar-head counterparts for efficient gene transfection [41]. Then, Maslov M. A.
synthesized novel cationic cholesteryl glucosides with different heterocyclic headgroups, and the
outcomes revealed that lipids/DNA complexes contained more fingerprint structures possessing
higher transfection activity both in the presence and in the absence of serum [42]. Apart from the
monosaccharide-based cationic glycolipids mentioned above, polysaccharide-based cationic lipids
were developed for nucleic acid delivery. Eliz Amar-Lewis furnished starch-based lipids and
applied them to delivery siRNA to NAR cells. The results reflected that these lipids showed high
cell uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers
governed the kinetics of siRNA transfection [17]. Chae S. Y., et al. developed deoxycholic
acid-conjugated chitosan oligosaccharide nanoparticles for transferring plasmid DNA to Hek 293
cells with high efficiency and low cytotoxicity. More than 20 and 100 times enhanced gene
transfections were observed by these nanoparticles mediated gene delivery than that of polylysine
[39-43]
(PLL) in the absence and presence of FBS, respectively [43]. The results from literatures
indicated that the sugar-based cationic glycolipids have high transfection efficiency, low toxicity
and higher biodegradability. Besides those merits reminded above, glycolipids can deliver gene to
target site by lectin-carbohydrate interactions [44-47]. Mariana Magalhães and coworkers developed
a glycolipid-based nanosystem incorporating a galactose, which can promote the nanosystem
interaction with the asialoglycoprotein receptor (ASGP-R) and finally increase the gene delivery
efficiency and specificity to HepG2 cells (human HCC) [44]. Based on similar principles, Ivanova
E. A. et al. furnished a bivalent galactose-containing neutral lipid system for targeted gene
delivery system to the hepatocytes, this lipid system showed high agglutination of cationic
liposomes by ricinus communis lectin (RCA120). Furthermore, lots of glycolipid-based targeting
gene delivery systems and the type of targeted cells determined by carbohydrate terminal of
glycolipid were comprehensive reviewed [46-47]
.
2

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DOI: 10.1039/C5OB02107C
Pioneering research indicates that specific deliver gene to tumor cells can be accomplished
by targeting cell surface lectins. However, different carbohydrate terminal of glycolipid only can
be recognized by specific lectin caused to target delivery. Therefore, we designed and synthesized
two novel series of cationic cyclic glycolipids containing quaternary ammonium headgroup and
varying hydrophobic alkyl tails for gene delivery, and to evaluate its efficacy and specificity. The
two novel series of cationic glycolipids were distinguished by hydrophobic tail attached to the
different positions of lipids. One serial (lipids 1-3, Scheme 1) was directly bonded to glucose in
ether, while the other serial (lipids 4-6, Scheme 1) was attached to the positively charged nitrogen
atoms. The size and zeta potential of lipids and complexes were determined by DLS, and the
ability of DNA-banding as well as the cytotoxicity of the lipids were measured via agarose gel
electrophoresis and MTT respectively. Furthermore, the morphology of the lipids and complexes
were analyzed by atomic force microscopy (AFM). All the lipids were applied to transfer pEGFP
into the HEK293 cells. The transfection efficiencies of those lipids were also assayed, and the
relationship between structure and activity was discussed.
2. Results and discussion
2.1. Synthesis of cationic glucolipids
Synthetic routes adopted for preparing the cationic glycolipids 1-3 and glycolipids 4-6 are
shown in Scheme 1 and Scheme 2 respectively. The key intermediate 3’-Azidopropyl
β-D-glucopyranoside (13) was prepared conventionally in five steps. Briefly, intermediate 9 was
synthesized by peracetylation of D-glucose (7) with acetic anhydride and perchloric acid and
followed by selective anomeric deacetylation with ammonia in methanol. Treated intermediate 9
with trichloroacetonitrile in the presence of potassium carbonate in anhydrous dichloromethane
provided the glycosyl donor 10. Glycosyl donor 10 was coupled with 3-chloro-1-propanol in
anhydrous dichloromethane catalyzed by TMSOTf to give β-glycoside 11. Then the key
intermediate 13 was furnished by azidation of compound 11 with sodium azide in
N,N-dimethylformamide and followed by deacetylation with ammonia in methanol. Intermediate
13 was selectively protected by isopropylidination to yield compound 14, which was transferred
into compound 15 by Williamson ether reaction with alkyl bromide in the presence of sodium
cyanide in anhydrous N,N-dimethylformamide. Treated compound 15 with 2% acetyl chloride in
methanol, compound 16 was afforded. Glycolipids 1-3 were then prepared by one-pot reduction of
azide to tertiary amine 17 followed by quaterisation reaction. Cationic glycolipids 4-6 were
synthesized from compound 13 by Staudinger reduction, followed tertiary amination with alkyl
bromines in the presence of potassium carbonate and quaterisation reaction with methyl iodine.
The structures of the important intermediates and lipids 1-6 were characterized and confirmed by
1H NMR, 13C NMR, gCOSY and HSQC. The detail procedure for synthesis of the target lipids (1
and 4) and NMR data were included in section 3.2 in this paper, the other lipids’ were introduced
in the supporting information.
Scheme 1: Synthesis of Cationic Glycolipids 1-3
3

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DOI: 10.1039/C5OB02107C
OAc
O
OAc
O
OAc
O
OH
O
a
b
c
d
AcO
AcO
AcO
AcO
AcO
AcO
HO
HO
OAc
OH
OH
NH
AcO
OAc
OAc
OH
OCCCl3
7
8
9
10
OAc
O
OAc
O
OH
O
e
f
g
AcO
AcO
AcO
AcO
HO
O
N3
O
N3
O
Cl
HO
AcO
AcO
HO
11
12
13
OH
O
O
O
h
i
j
O
O
O
O
HO
RO
O
N3
O
N3
O
N3
HO
RO
HO
OH
RO
14
15 a-c
RO 16 a-c
OH
18 a
18 b
18 c
1
2
3
a: R =n-C12H25
b: R =n-C14H29
c: R =n-C16H33
,
,
,
k
Lipid
Lipid
Lipid
O
O
HO
RO
HO
RO
O
N
O
N
I-
RO
RO
17 a-c
o
Reagents and conditions: (a) Ac2O, HClO4, 0-20 C, 10 h, 94.8%; (b) NH3(g), CH3OH,
1,4-Dioxane, 1 h, 73.9%; (c) CCl3CN, K2CO3, DCM, 12 h, 85.1%; (d) HOCH2CH2CH2Cl,
TMSOTf, DCM, -20 oC to rt, 4 h, 50.7%; (e) NaN3, DMF, 75 oC, 10 h, 84.9%; (f) NH3(g), CH3OH,
9 h, 95.0%; (g) H3CC(OCH3)2CH3, concd H2SO4, 6 h, 46.3%; (h) Alkyl Bromide, NaH, DMF, 24
h, 41.5%; (i) CH3COCl, CH3OH, 2 h, 61.7%; (j) H2, 5% Pd/C, HCHO, CH3OH, 24 h, 48.8%; (k)
CH3I, THF, 20 h, 32.5%.
Scheme 2: Synthesis of Cationic Glycolipids 4-6
OH
O
OH
O
OH
O
R
N
R
N
a
b
C
HO
HO
HO
HO
O
NH2
HO
HO
O
R
O
R
I-
13
HO
19
HO
HO
20a-c
21a
21b
21c
1
2
3
a: R =n-C12H25
b: R =n-C14H29
c: R =n-C16H33
,
,
,
Lipid
Lipid
Lipid
Reagents and conditions: (a) PPh3, H2O, THF, reflux, 5 h, 85.4%.; (b) Alkyl Bromide, CH3OH,
CH3CH2OH, reflux, 48 h, 41.3%; (c) CH3I, THF, 20 h, 86.8%.
2.2. In vitro transfection biology
High transfection efficiency is a prerequisite for an applicable non-viral gene vector. To
investigate the gene transfection efficiencies of the synthesized glycolipids, the fluorescence
microscopy assay was carried out using pEGFP-C3 plasmid DNA as the reporter gene in HEK293
cells, and lipo2000 was used as the positive control. Five levels of N/P ratios (2, 4, 6, 8 and 10)
were set for each lipid and corresponding enhanced green fluorescent protein expression in
HEK293 cells were observed by an inverted fluorescent microscope. The highest density of
transfected cells for each glycolipid was obtained and corresponding fluorescence image was
shown in Fig. 1. The fluorescence microscope images of HEK293 cells transfected by
glycolipid/DNA complexes in the other four levels of N/P ratios were displayed in supporting
information (Figure S1). The glycolipids 1-2 (Fig. 1, B and C) with the hydrophobic tails linked to
glucose cycle directly in ether were found to be inefficient in transfecting HEK293 cells compared
to lipo2000 (Fig. 1, A). The transfection efficiency of glycolipids 5-6 with the hydrophobic tails in
the positively charged nitrogen atoms (Fig. 1, F and G) were as good as or better than lipo2000.
However, glycolipid 3 and 4 (Fig. 1, D, E; Figure S1) did not have any transfection efficiency at
all N/P ratios. In order to reflect the transfection efficiency between lipo2000 and lipids 5-6
accurately, the cell counting of lipo2000 (2 μL) and lipids 5-6 (N / P = 4, 6, 8, 10) in HEK293
4

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Organic & Biomolecular Chemistry
cells were shown in Fig. 2. As shown in Fig. 2, the transfection efficiency of lipid 5 and 6 werVeiew Article Online
DOI: 10.1039/C5OB02107C
increased with the lipid/DNA charge ratios from 4 to 10, and they got the maximum efficiency at
N/P 10, 8 respectively, which were better than lipo2000. Obviously, the lipid 6 with double C16
saturated chains was more efficient than lipid 5 with double C14 saturated chains. In a word, the
transfection results indicated that the position and the length of the hydrophobic tails in the
glucose-based lipids had a significant influence on transfection efficiency.
A
B
C
D
E
F
G
Fig. 1. Fluorescence microscope images of HEK293 cells transfected by glycolipid/DNA
complexes. A: lipo2000 (2 μL), B: lipid 1 (N/P=10), C: lipid 2 (N/P=8), D: lipid 3 (N/P=8), E:
lipid 4 (N/P=6), F: lipid 5 (N/P=8), G: lipid 6 (N/P=8).
160
5
6
lipo2000
140
120
100
80
60
40
20
0
4
6
8
10
12
Lipo 2000
N/P
Fig 2. In vitro gene delivery efficiencies of lipids 5-6 in HEK293 cells at various glycolipid/DNA
charge ratios (4:1 - 10:1). Lipo2000 (2 μL) was used as the positive control. (p<0.05)
Besides HEK 293 cells, we transfected other three cells including Hela, SW480 and HepG2
5

Organic & Biomolecular Chemistry
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to further investigate the different transfection performances of the designed glycolipids. As thVeiew Article Online
DOI: 10.1039/C5OB02107C
fluorescence microscope images shown in supporting information (Figure S2), Lipid 2 showed
very weak gene expression in Hela cells at all the levels of N/P ration. However, lipid 3 showed
gradually decreased transfection efficiency in Hela cells with the increase of N/P rations. Lipid 5
and 6 displayed relatively much lower transfection efficiency in HepG2 cells and SW480 cells
respectively. But lipid 6 showed slightly enhanced transfection efficiency in HepG2 cells
compared to lipid 5. The transfection efficiency of the designed glycolipids in above-mentioned
three cells was different and relatively lower than HEK 293 cells, which was due to the fact that
the in vitro transfection capabilities are cell-line-dependent and the transfection efficiency of
nonviral vectors depends on various parameters such as cytotoxicity, DNA condensation and
protection, serum stability, cellular uptake efficiency, or intracellular trafficking.
2.3. Sizes and zeta potentials (ζ)
Physicochemical properties of lipids and their complexes with nucleic acids are very
important characteristics that can influence the transfection efficiency. The sizes and surface
potentials of net lipids and complexes formed with pEGFP-C3 at N/P charge ratios (2 : 1, 4 : 1, 6 :
1, 8 : 1, 10 : 1) were evaluated by using the dynamic light scattering (DLS). As shown in table 1
and table 2, the average sizes of lipids 1-6 and their complexes were nano-sized particles smaller
than 250 nm, which had a potential for transferring nucleic acids into the cells in the absence of
serum [48]. The sizes of lipids 1-6 were smaller than 100 nm except lipid 4 (247 nm). After
lipids/DNA complexes were formed, the sizes of the complexes formed from lipids 1-2 were not
significantly changed. However, the sizes of lipids 3-4/DNA complexes decreased and lipids
5-6/DNA complexes increased compared with the corresponding net lipids. Size distribution also
became little narrower after complexation. As shown in Fig. 1., the lipids 5-6 showed higher
transfer efficiency than other lipids. This may be attributed to their larger sizes, because large
particles taken up by cells lead to the formation of large intracellular vesicles, which disrupt and
release DNA into the cytoplasm more easily [49]. Though the lipid 4/DNA complex had a large size,
it had low transfection efficiency probably owing to its high cell toxicity (Fig. 5). For the
interaction between lipids and DNA, lipoplexes and anionic cell membranes are electrostatic; the
positive charged complexes could encourage electrostatic interaction with the anionic plasma
membrane for sufficient cellular uptake. So Zeta potential was an important factor in
characterizing the surface charges of liposomes and lipoplexes. As shown in table 1, 2, all
glycolipids and lipids/DNA complexes exhibited positive zeta potentials. The zeta potentials of
lipids 1-6 were 35-70 mV and their complexes decreased to 7-40 mV after DNA complexation.
Table 1. The average size and zeta potential of lipids 1-6.
Glycolipids
lipid 1
Zeta potential (mV)
+46.0 ± 0.5
Average size (nm)
90.3 ± 8.5
PDI
0.318 ± 0.021
0.501 ±0.080
0.466 ± 0.074
0.333 ± 0.032
0.429 ± 0.056
0.388 ± 0.041
lipid 2
+36.2 ± 0.6
63.4 ± 10.1
109.7 ± 9.4
247.1 ± 8.7
80.4 ± 9.0
lipid 3
+37.4 ± 0.4
lipid 4
+69.5 ± 0.3
lipid 5
+42.5 ± 0.5
lipid 6
+40.0 ± 0.4
75.0 ± 8.8
Table 2. The average size and zeta potential of lipids/DNA complexes.
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PDDIOI: 10.1039/C5OB02107C
Lipids/DNA complexes N/P ratios Zeta potential (mV) Average size (nm)
2:1
4:1
6:1
8:1
10:1
2:1
4:1
6:1
8:1
10:1
2:1
4:1
6:1
8:1
10:1
2:1
4:1
6:1
8:1
10:1
2:1
4:1
6:1
8:1
10:1
2:1
4:1
6:1
8:1
10:1
+11.2 ± 1.2
+13.5 ± 1.3
+19.9 ± 2.2
+15.9 ± 3.3
+15.9 ± 1.3
+14.5 ± 2.7
+18.8 ± 1.9
+16.7 ± 1.4
+7.1 ± 1.6
+16.2 ± 1.5
+10.8 ± 3.8
+24.8 ± 3.3
+26.6 ± 2.2
+20.8 ± 1.6
+29.1 ± 2.1
+15.9 ± 1.6
+17.4 ± 2.2
+41.2 ± 2.4
+30.0 ± 3.9
+21.7 ± 4.7
+12.7 ± 2.5
+15.3 ± 2.8
+18.4 ± 1.8
+15.7 ± 2.7
+27.2 ± 1.7
+10.2 ± 3.7
+17.9 ± 3.5
+24.7 ± 2.9
+32.8 ± 3.3
+29.2 ± 3.5
130.9 ± 4.9
133.0 ± 0.8
136.5 ± 7.2
123.3 ± 5.7
91.4 ± 8.2
144.9 ± 2.8
86.1 ± 1.9
86.1 ± 5.1
0.507 ± 0.054
0.487 ± 0.047
0.565 ± 0.058
0.597 ± 0.063
0.217 ± 0.049
0.368 ± 0.087
0.323 ± 0.071
0.377 ± 0.082
0.341 ± 0.064
0.367 ± 0.078
0.557 ± 0.091
0.482 ± 0.052
0.534 ± 0.088
0.603 ± 0.097
0.472 ± 0.085
0.490 ± 0.079
0.470 ± 0.065
0.320 ± 0.050
0.425 ± 0.066
0.313 ± 0.054
0.417 ± 0.068
0.533 ± 0.081
0.505 ± 0.093
0.599 ± 0.086
0.526 ± 0.070
0.280 ± 0.058
0.308 ± 0.046
0.407 ± 0.083
0.284 ± 0.037
0.173 ± 0.028
lipid 1/DNA
lipid 2/DNA
lipid 3/DNA
lipid 4/DNA
lipid 5/DNA
lipid 6/DNA
66.4 ± 9.0
77.1 ± 2.3
173.3 ± 23.5
148.5 ± 16.2
140.3 ± 15.3
92.8 ± 11.5
115.7 ± 11.5
190.3 ± 16.7
188.4 ± 9.7
173.0 ± 9.3
121.2 ± 14.3
114.1 ± 10.5
147.3 ± 16.9
176.0 ± 10.4
194.6 ± 24.5
183.3 ± 20.6
147.2 ± 10.8
186.0 ± 4.5
207.9 ± 10.6
249.0 ± 16.4
129.0 ± 8.4
167.2 ± 3.8
2.4 Atomic force microscopy (AFM)
Further characterization was done in order to reach a better understanding of the morphology
of the lipids and complexes, which were analyzed by atomic force microscopy (AFM). As
opposed to DLS, the AFM scanning was done on a dried mica sheet. Although the dried form of
the complexes in AFM does not give an accurate information about the complexes' size as in a
suspension (as was done in DLS), it can give visual insight into the lipids' and complexes'
morphology and size. Firstly, the samples of lipids 2 and 6 were scanned in order to visualize the
glycolipids before complexation. It seemed that the lipid 2 spread well on the mica and irregular
spherical particles were formed with a diameter of 100-200 nm (Fig. 3. A). However, lipid 6
formed spherical particles (about 200 nm) or aggregated particles (>400 nm) (Fig. 3. C).
Lipoplexes formed at N/P ratio of 8 : 1 can be observed in a regular spherical form with its typical
diameter of approximately 50-200 nm (Fig. 3. B and D). AFM images also indicated lipoplexes
are much better dispersed than the net lipids.
A
B
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DOI: 10.1039/C5OB02107C
C
D
Fig. 3. Atomic Force Microscopy (AFM) images of lipid 2 (50 µmol/L) (A), lipid 2/DNA complex
at N/P=8 (80 µmol/L) (B), lipid 6 (100 µmol/L) (C), and lipid 6/DNA complex at N/P=8 (100
µmol/L) (D).
2.5 Lipid : DNA binding assay
DNA binding affinity was considered as an important parameter in the formation of
lipids/DNA lipoplexes. Herein, DNA binding affinities of glycolipids were evaluated by agarose
gel retardation assay. As shown in Fig. 4, the intensity of free DNA migrating bands gradually
decreased with the addition of the percentage of glycolipids. It could be seen that DNA was
completely retarded with the addition of lipids 1-3 at N/P 1-2, while weaker DNA retardation of
lipids 4-6 was observed at N/P 2-3. The results indicated that the lipids 1-3 with the hydrophobic
tails linked to glucose cycle directly in ether possessed higher DNA binding affinity than that of
lipids 4-6 with the hydrophobic tails in the positively charged nitrogen atoms, and the high DNA
binding capability could be attributed to the combination effect of the electrostatic and
hydrophobic interactions. Although the lipids 1-3 had a better capacity for DNA-binding
relative to lipid 4-6, the disparity was little. In short, the glycolipids had an excellent capability for
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DNA-binding, because all of them could combine with DNA completely at low N/P ratioDsO(2I: 1o0r.1033)9./C5OB02107C
DNA
1
2
DNA
1
2
DNA
1
2
0.3
0.5
3
0.3
0.5
3
0.3
0.5
3
A
B
C
DNA
1
2
DNA
1
2
DNA
1
2
0.3
0.5
3
0.3
0.5
3
0.3
0.5
3
D
E
F
Fig. 4. Electrophoretic gel patterns for lipoplex-associated DNA in gel retardation assay. (A)–(F)
refer to lipids 1-6, respectively. The lipid/DNA charge ratios are indicated at the top of each lane.
The details of the treatment are as described in the text.
2.6 Toxicity studies
Cytotoxicity was considered as a key issue in gene delivery, because favorable gene carriers
should be low cytotoxic. Thus, the correlations between molecular structure and cytotoxicity of
cationic lipids were important to elucidate. MTT-based cell viabilities of lipids 1-6 were evaluated
in HEK293 cells used for in vitro transfection across the entire range of lipid/DNA charge ratios
used in the actual transfection experiments and commercially available lipo2000 was utilized as
the positive control. As shown in Fig. 5, the cells incubated with lipids 1-3 showed higher cell
viabilities (95-140%) than that of lipids 4-6 (50-110%) within the N/P ratios from 2 to 10. The
result indicated that the lipids with the hydrophobic tails in the positively charged nitrogen atoms
had more cytotoxicity related to the lipids with the hydrophobic tails linked to glucose cycle
directly in ether. Especially, the lipid 4 with dodecyl hydrophobic chains had the highest cell
toxicity among all the lipids, and its cell viability decreased with the increase of N/P ratios and
merely half part of lipo2000 when the N/P ratio reached to 10:1. According to the Fig. 1, the lipid
4 did not have any transfection efficiency possibly owing to its lowest cell viability. However, the
cell viabilities of lipids 5-6, which had the highest transfection efficiency, increased with the
lengthening of the hydrophobic chains, closed to the lipo2000.
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1
4
2
5
3
6
160
140
120
100
80
Lipo2000
60
40
20
0
2
4
6
8
10
12
Lipo 2000
N/P
Fig. 5. Representative percent cell viabilities of lipids 1-6 in HEK293 cells using MTT assay.
The toxicity assays were performed as described in the text. The data presented are the average
values of three independent experiments (n = 3, p<0.05).
3. Materials and methods
3.1 Materials
Lauryl bromide, Myristyl bromide, Cetyl bromide, 2,2-Dimethoxypropane, 33% aqueous
Formaldehyde, trimethylsilyl trifluoromethanesulfonate(TMSOTf), Sodium azide, 5%Pd/C,
Trichloroacetonitrile and 1-Chloro-3-hydroxypropane were purchased from Shanghai Bangcheng
Chemical Co. Ltd. Sodium cyanide was purchased from Tianjing Chemical Reagent Resarch
Institute. Glucose was purchased from sinoparm Chemical Reagent Co. Ltd. Triphenylphosphine
was purchased from Hunan Huihong Chemical Regent Co. Ltd. Iodomethane was purchased from
Xiya Reagent. Column chromatography was performed using 200-300 mesh silica gel.
Biowest agarose was purchased from Gene Tech, Shanghai. Lipofectamine 2000 was
received from Invitrogene Life Technology, USA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) was obtained from Amresco. Penicillin-Streptomycin Solution and
Dulbecco’s Modified Eagle Media (DMEM) was purchased from HyClone. Trypsin and fetal
bovine serum (FBS) were purchased from the Gbico. 96-well and 24-well cultivation plates and
50 mL cell cultivation flasks were purchased from Corning Co. Ltd., USA. Plasmid DNA and cells
line were kindly provided by the lab of Prof. Shuanglin Xiang, School of Life Sciences, Hunan
Normal University. In this work, all the other reagents and solvents were analytical grade and
were utilized without further purification. 1H NMR, gCOSY and HSQC spectra were recorded on
a Bruker 500MHz and 13C NMR spectra were recorded on a Bruker 125MHz. Electrospray
ionization mass spectrometry (ESI-MS) in positive and negative mode was performed on a
10

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Organic & Biomolecular Chemistry
Finnigan LCQ Advantage (Thermo Finnigan LCQ) equipped with an atmosphericDOpIr:e1s0s.1u0r3Ve9ie/Cw5AOrtiBc0le2O1n0l7inCe
ionization (API) source.
3.2 Syntheses of cationic lipids 1-6.
As representative details, syntheses and spectral characterization of lipids 1 and 4 and all
their synthetic intermediates shown in Schemes 1 and Schemes 2 were provided. Lipids 2-3 and
lipids 5-6 were synthesized following essentially the same protocols adopted for preparing lipids 1
and 4, respectively. The detailed procedure for synthesis of the target lipids 2, 3, 5, 6, NMR datas
and NMR spectrums were included in the supporting information.
3.2.1 Synthesis of Cationic Glycolipids 1 (Scheme 1)
1,2,3,4,6-Penta-O-acetyl-α,β-D-glucopyranose (8)
The acetic anhydride (393.5 mL, 4.2 mol) in 1.0 L round flask was cooled to 0oC under
stirring. Then perchloric acid (2.0 mL) was added dropwise. The D-glucose (7, 100.0 g, 55.5
mmol) was added in partition under the temperature was not over 20oC. The reaction mixture was
stirring until TLC (petroleum ether : ethyl acetate = 2 : 1) showed the starting material was
disappeared, during which time the temperature was gradually raised to ambient temperature.
DCM (500.0 mL) was added, and the mixture was put into a flask with ice water. The mixture was
dispensed, and the organic layer was dried by anhydrous Na2SO4 and concentrated to dryness. The
residue was recrystallized with petroleum ether-ethyl acetate to give compound 8 as a white solid
(205.4 g, 94.8%).
2,3,4,6-Tetra-O-acetyl-α,β-D-glucopyranose (9)
Ammolonia was bubbled to the solution of compound 8 (50.0 g, 128.2 mmol) in methanol /
1,4-dioxane (250.0 mL, Vmethanol : Vdioxane = 2 : 5). The reaction mixture was stirring until TLC
(petroleum ether : ethyl acetate = 2 : 1) showed the starting material was almost disappeared. Then
the mixture was concentrated and the residue was purified by silica gel column chromatography
with petroleum ether : EtOAc = 2 : 1 as the eluent to give compound 9 (33.0 g, 73.9%) as a syrup.
2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate (10)
Anhydrous K2CO3 (14.0 g, 101.4 mmol) and trichloroacetonitrile (22.6 mL, 225.4 mmol)
were added to the solution of compound 9 (33.0 g, 94.7 mmol) in dry DCM (200.0 mL). The
mixture was stirring at room temperature until TLC (petroleum ether : ethyl acetate = 3 : 1)
showed the starting material was disappeared. The mixture was filtered and concentrated to
dryness. The residue was purified by silica gel column chromatography with petroleum ether :
EtOAc = 2 : 1 as the eluent to give compound 10 (39.7 g, 85.1%) as a syrup.
3’-Chloropropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (11)
The compound 10 (32.0 g, 65.0 mmol) and 4Ǻ MS (2.0 g) were dried at 60 oC under vacuum
for 2 h, then 3-chloro-1-propanol (13.0 mL, 155.5 mmol) and dry DCM (250.0 mL) were added.
The mixture was stirred and cooled to -20oC, and TMSOTf (106.0 µL, 0.6 mmol) was added
dropwise. The mixture was stirred for 2 h, during which time the temperature was gradually raised
to ambient temperature. The mixture was quenched with Et3N (0.2 mL) and washed with water
and extracted with DCM for three times. The organic layer was dried by anhydrous Na2SO4 and
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concentrated to dryness. The residue was purified by silica gel column chromatography with
petroleum ether : EtOAc = 4 : 1 as the eluent to give a syrup, and the syrup recrystallized with
petroleum ether and ethyl acetate to give compound 11 (14.0 g, 50.7%) as a white solid. 1H NMR
(500 MHz, CDCl3): δ (ppm): 5.19 (dd, 1 H, J2,3 = J3,4 = 9.5 Hz, H-3), 5.06 (dd, 1 H, J4,3 = J4,5 = 9.5
Hz, H-4), 4.95 (dd, 1 H, J2,1 = 8.0 Hz, J2,3 = 9.5 Hz, H-2), 4.90 (d, 1 H, J1,2 = 8.0 Hz, H-1), 4.25 (dd,
1 H, J6a,5 = 3.5 Hz, J6a,6b = 12.5 Hz, H-6a), 4.13 (dd, 1 H, J6b,5 = 2.5 Hz, J6a,6b = 12.5 Hz, H-6a),
3.99-3.95 (m, 1 H, OCH2CH2CHHCl), 3.70-3.65 (m, 2 H, H-5, OCH2CH2CHHCl), 3.59-3.56 (m,
2 H, OCH2CH2CH2Cl), 2.06 (s, 3 H, CH3CO), 2.03 (s, 3 H, CH3CO), 2.00 (s, 3 H, CH3CO), 1.98
(s, 3 H, CH3CO), 1.97-1.88 (m, 2 H, OCH2CH2CH2Cl); 13C NMR (125 MHz, CDCl3): δ (ppm)
170.6 (1 C, CH3CO), 170.2 (1 C, CH3CO), 169.4, 169.4 (2 C, 2 CH3CO), 101.0 (1 C, C-1), 72.7 (1
C, C-3), 71.8 (1 C, C-2), 71.3 (1 C, C-5), 68.4 (1 C, C-4), 66.4 (1 C, OCH2CH2CH2Cl), 61.9 (1 C,
C6), 41.3 (1 C, OCH2CH2CH2Cl), 32.2 (1 C, OCH2CH2CH2Cl), 20.7(1 C, CH3CO), 20.6(1 C,
CH3CO), 20.5,20.5 (2 C, CH3CO).
3’-Azidopropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (12)
The mixture of compound 11 (3.0 g, 7.1 mmol), dry DMF (5.0 mL), and NaN3 (2.8 g, 42.6
mmol) was stirred at 75oC until TLC (petroleum ether : ethyl acetate = 4 : 1) showed the starting
material was disappeared. Then the reaction mixture was filtered, and the filter was diluted with
DCM (150.0 mL), washed with water and dried over anhydrous Na2SO4. The mixture was filtered
and concentrated in vacuo to dyness. The residue was purified by silica gel column
chromatography with petroleum ether : EtOAc = 4 : 1 as the eluent to give compound 12 (2.6 g,
84.9%) as a yellow oil. 1H NMR (500 MHz, CDCl3): δ (ppm): 5.19 (dd, 1 H, J2,3 = J3,4 = 9.5 Hz,
H-3), 5.07 (dd, 1 H, J4,3 = J4,5 = 9.5 Hz, H-4), 4.97 (dd, 1 H, J2,1 = 8.0 Hz, J2,3 = 9.5 Hz, H-2), 4.90
(d, 1 H, J1,2 = 8.0 Hz, H-1), 4.25 (dd, 1 H, J6a,5 = 4.5 Hz, J6a,6b = 12.5 Hz, H-6a), 4.14 (dd, 1 H, J6b,5
= 3.6 Hz, J6a,6b = 12.5 Hz, H-6a), 3.94-3.92 (m, 1 H, OCH2CH2CHHN3), 3.70-3.67 (m, 1 H,
OCH2CH2CHHN3), 3.61-3.58 (ddd, 1 H, J5,4 = 9.5 Hz, J5,6a = 4.5 Hz, J5,6b = 3.6 Hz, H-5),
3.34-3.37 (m, 2 H, OCH2CH2CH2Cl), 2.07 (s, 3 H, CH3CO), 2.04 (s, 3 H, CH3CO), 2.01 (s, 3 H,
CH3CO), 1.99 (s, 3 H, CH3CO), 1.80-1.86 (m, 2 H, OCH2CH2CH2Cl); 13C NMR (125 MHz,
CDCl3): δ (ppm) 170.6 (1 C, CH3CO), 170.2 (1 C, CH3CO), 169.3 (1 C, CH3CO), 169.2, 169.2 (2
C, CH3CO), 100.8 (1 C, C-1), 72.8 (1 C, C-3), 71.8 (1 C, C-2), 71.3 (1 C, C-5), 68.4 (1 C, C-4),
66.4 (1 C, OCH2CH2CH2Cl), 61.9 (1 C, C-6), 47.9 (1 C, OCH2CH2CH2Cl), 28.9 (1 C,
OCH2CH2CH2Cl), 20.7 (1 C, CH3CO), 20.6 (1 C, CH3CO), 20.5, 20.5 (2 C, CH3CO).
3’-Azidopropyl β-D-glucopyranoside (13)
Ammonia was bubbled to the solution of compound 12 (3.0 g, 7.1 mmol) in methanol (20.0
mL). The reaction mixture was stirring until TLC (petroleum ether : ethyl acetate = 2 : 1) showed
the starting material disappeared. Then the mixture was concentrated and the residue was purified
by silica gel column chromatography with petroleum ether : EtOAc = 2 : 1 as the eluent to give
compound 13 (1.5 g, 95.0%) as a syrup.
3’-Azidopropyl 4,6-O-isopropylidene-β-D-glucopyranoside (14)
The mixture of compound 13 (1.5 g, 5.7 mmol) and 2,2-dimethoxypropane (10 mL, 81.3
mmol) was stirred at the room temperature, and four drops of conc. sulfuric acid was added. The
reaction mixture was stirring until TLC (petroleum ether : ethyl acetate = 3 : 1) showed the
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starting material disappeared. The mixture was neutralized with K2CO3, filtered and evaporated to
dryness. The residue was purified by silica gel column chromatography with petroleum ether :
EtOAc = 3 : 1 as the eluent to give compound 14 (0.8 g, 46.3%) as a syrup. 1H NMR (500 MHz,
CDCl3): δ (ppm): 4.34 (d, 1 H, J1,2 = 7.5 Hz, H-1), 3.98-3.90 (m, 2 H, H-6a, OCH2CH2CHHN3),
3.78 (dd, 1 H, J6b,5 = 5.4 Hz, J6a,6b = 10.5 Hz, H-6a), 3.68-3.64 (m, 2 H, H-3, OCH2CH2CHHN3),
3.57 (dd, 1 H, J4,3 = J4,5 = 9.5 Hz, H-4), 3.46-3.41 (m, 2 H, H-2, OCH2CH2CH2N3), 3.27 (ddd, 1 H,
J
5,4 = 9.5 Hz, J5,6a = 4.5 Hz, J5,6b = 5.4 Hz, H-5), 1.91-1.86 (m, 2 H, OCH2CH2CH2N3), 1.50 (s, 3 H,
C(CH3)2), 1.43 (s, 3 H, C(CH3)2); 13C NMR (125 MHz, CDCl3): δ (ppm) 103.2 (1 C, C-1), 99.8 (1
C, C(CH3)2), 74.6 (1 C, C-2), 73.5 (1 C, C-3), 73.0 (1 C, C-4), 67.3 (1 C, C-5), 67.0 (1 C, C-6),
62.0 (1 C, OCH2CH2CH2N3), 48.3 (1 C, OCH2CH2CH2N3), 29.0 (1 C, OCH2CH2CH2N3), 28.9,
19.0 (2 C, C(CH3)2).
3’-Azidopropyl 2,3-di-O-dodecyl-4,6-O-isopropylidene-β-D-glucopyranoside (15a)
NaH (100.0 mg, 4.0 mmol) was added slowly to the solution of compound 14 (0.2 g, 0.65
mmol) in dry dimethylformamide (2.0 mL) and then 1-bromododecane (0.65 mL, 2.6 mmol) was
added dropwise. The reaction mixture was stirring until TLC (petroleum ether : ethyl acetate = 2 :
1) showed no change occurred. The mixture was diluted with DCM (10 mL), and washed with
water for three times. The organic layer was dried by anhydrous Na2SO4 and concentrated to
dryness. The residue was purified by silica gel column chromatography with petroleum ether :
EtOAc = 16 : 1 as the eluent to give compound 15a (170.0 mg, 41.5 %) as a syrup. 1H NMR
(500MHz, CDCl3): δ (ppm) 4.30 (d, 1 H, J1,2 = 7.5 Hz, H-1), 3.93-3.86 (m, 2 H, OCH2CH2CHHN3,
H-6a), 3.75-3.70 (m, 3 H, OCH2(CH2)10CH3, H-6b), 3.66-3.58 (m, 3 H, OCH2CH2CHHN3,
OCH2(CH2)10CH3), 3.53 (dd, 1 H, J4,3 = 8.5 Hz, J4,5 = 9.5 Hz, H-4), 3.89-3.41 (m, 2 H,
OCH2CH2CH2N3), 3.25 (dd, 1 H, J3,2 = 9.0 Hz, J3,4 = 8.5 Hz, H-3), 3.16 (ddd,1 H, J5,4 = 9.5 Hz,
J5,6a = 5.5 Hz, J5,6b = 3.6 Hz, H-5), 3.06 (dd, 1 H, J2,1 = 7.5 Hz, J2,3 = 9.0 Hz, H-2), 1.87-1.84 (m, 2
H, OCH2CH2CH2N3), 1.55-1.51 (m, 4 H, 2 OCH2CH2(CH2)9CH3), 1.46 (s, 3 H, C(CH3)2), 1.38 (s,
3 H, C(CH3)2), 1.38-1.20 (m, 36 H, 2 OCH2CH2(CH2)9CH3), 0.88 (t, 6 H, J = 6.5 Hz, 2
OCH2CH2(CH2)9CH3); 13C NMR (125 MHz, CDCl3): δ (ppm) 104.0 (1 C, C-1), 99.2 (1 C,
C(CH3)2), 82.2 (1 C, C-2), 81.7 (1 C, C-3), 73.9 (1 C, C-4), 73.6 (1 C, OCH2(CH2)10CH3), 73.2 (1
C, OCH2(CH2)10CH3), 66.9 (1 C, C-5), 66.7 (1 C, OCH2CH2CH2N3), 62.2 (1 C, C-6), 48.2 (2 C,
OCH2CH2CH2N3), 31.9, 30.3, 30.2, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 26.4 (21 C, some signals
were overlapped, 2 OCH2(CH2)10CH3, OCH2CH2CH2N3), 22.6 (1 C, C(CH3)2), 19.0 (1 C,
C(CH3)2), 14.0, 14.0 (2 C, 2OCH2CH2(CH2)9CH3).
3’-Azidopropyl 2,3-di-O-dodecyl-β-D-glucopyranoside (16a)
The mixture of compound 15a (170.0 mg, 0.27 mmol) and dry methanol (10.0 mL) was
cooled to 0oC under stirring, and then the acetyl chloride (0.2 mL, 2.4 mmol) was added dropwise.
The reaction mixture was stirring until TLC (petroleum ether : ethyl acetate = 2 : 1) showed the
starting material disappeared, during which time the temperature was gradually raised to ambient
temperature. The mixture was evaporated to dryness and the residue was purified by silica gel
column chromatography with petroleum ether : EtOAc = 2 : 1 as the eluent to give compound 16a
(100.0 mg, 61.7%) as a white oil. 1H NMR (500 MHz, CDCl3): δ (ppm) 4.30 (d, 1 H, J1,2 = 8.0 Hz,
H-1), 3.96-3.87 (m, 1 H, OCH2CH2CHHN3), 3.88-3.87 (m, 2 H, OCHH(CH2)10CH3, H-6a),
3.79-3.73 (m, 2 H, OCHH (CH2)10CH3, H-6b), 3.64-3.56 (m, 3 H, OCH2(CH2)10CH3,
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OCH2CH2CHHN3), 3.47-3.40 (m, 3 H, OCH2CH2CH2N3, H-4), 3.34-3.28 (m, 1 H, H-5), 3.19
(dd,1 H, J3,2 = J3,4 = 9.0 Hz, H-3), 3.06 (dd, 1 H, J2,1 = 8.0 Hz, J2,3 = 9.0 Hz, H-2), 1.88-1.85 (m, 2
H, OCH2CH2CH2N3), 1.57-1.55 (m, 4 H, 2 OCH2CH2(CH2)9CH3), 1.24 (m, 36 H, 2
OCH2CH2(CH2)9CH3), 0.87 (t, 6 H, J = 6.5 Hz, 2 OCH2CH2(CH2)9CH3); 13C NMR (125 MHz,
CDCl3): δ (ppm) 103.7 (1 C, C-1), 84.2 (1 C, C-2), 82.1 (1 C, C-3), 74.8 (1 C, C-4), 73.6 (1 C,
OCH2(CH2)10CH3), 73.0 (1 C, OCH2(CH2)10 CH3), 70.4 (1 C, C-5), 66.7 (1 C, OCH2CH2CH2N3),
62.8 (1 C, C-6), 48.2 (2 C, OCH2CH2CH2N3), 31.9, 30.4, 30.3, 29.6, 29.5, 29.3, 29.2, 26.2, 26.1,
22.7 (21 C, some signals were overlapped, 2 OCH2(CH2)10CH3, OCH2CH2CH2N3), 14.1, 14.1 (2 C,
2 OCH2CH2(CH2)9CH3).
3’- (N,N-Dimethylamino)propyl 2,3-di-O-dodecyl-β-D-glucopyranoside (17a)
The mixture of compound 16a (1.0 g, 1.7 mmol) and formaldehyde (36%, 1.1 mL, 13.6
mmol), Pd/C (5%, 300 mg), methanol (60.0 mL) was stirred at the room temperature under H2
atmosphere. The reaction mixture was stirring until TLC (ethyl acetate : methanol = 5 : 2) showed
the starting material disappeared. The mixture was filtered and the filtrate was evaporated to
dryness. The residue was purified by silica gel column chromatography with EtOAc : methanol =
5 : 1 as the eluent to give compound 17a (0.5 g, 48.8%) as a colorless oil.
3’-(N,N,N-Trimethylaminonium iodine)propyl 2,3-di-O-dodecyl-β-D-glucopyranoside (18a,
lipid 1)
The mixture of compound 17a (240.0 mg, 0.4 mmol) and iodomethane (48.0 µL, 1.6 mmol),
THF (3.0 mL) was stirred at the room temperature. The reaction mixture was stirring until TLC
(ethyl acetate : methanol = 2 : 1) showed the starting material disappeared. The reaction mixture
was cooled with ice bath and a solid was precipitated. The mixture was filtered, and the filter cake
was washed with acetone (5.0 mL × 3) and dried by vacuum to give white solid lipid 1 (100.0 mg,
32.5%).1H NMR (500 MHz, CDCl3): δ (ppm) 4.31 (d, 1 H, J1,2 = 8.0 Hz, H-1), 3.92-3.85 (m, 4 H,
OCH2CH2CH2N(CH3)3, H-6a, OH), 3.78-3.64 (m, 6 H, OCHH(CH2)10CH3, OCH2(CH2)10CH3,
OCH2CH2CH2N(CH3)3, H-6b), 3.57-3.52 (m, 1 H, OCHH(CH2)10CH3), 3.46-3.32 (m, 11 H, OCH2
CH2CH2N(CH3)3, H-4, H-5), 3.25 (s,1 H, OH), 3.19 (dd, 1 H, J3,2 = 8.5 Hz , J3,4 = 8.0 Hz, H-3),
3.01 (dd, 1 H, J2,1 = 8.0 Hz, J2,3 = 8.5 Hz, H-2), 2.27-2.19 (m, 1 H, OCH2CHHCH2N(CH3)3),
2.06-2.02 (m, 1 H, OCH2CHHCH2N(CH3)3), 1.56-1.48 (m, 4 H, 2 OCH2CH2(CH2)9CH3), 1.24 (m,
36 H, 2 OCH2CH2(CH2)9CH3), 0.87 (t, 6 H, J = 7.0 Hz, 2 OCH2CH2(CH2)9CH3) ; 13C NMR (125
MHz, CDCl3): δ (ppm) 103.8 (1 C, C-1), 84.1 (1 C, C-3), 81.8 (1 C, C-2), 75.6 (1 C, C-5), 73.6 (1
C, OCH2(CH2)10CH3), 73.0 (1 C, OCH2(CH2)10CH3), 79.9 (1 C, C-4), 66.5 (1 C,
OCH2CH2CH2N(CH3)3), 64.7 (1 C, OCH2CH2CH2N(CH3)3), 61.4 (1 C, C-6), 53.9, 59.9, 53.9 (3 C,
OCH2CH2CH2N(CH3)3), 31.8, 30.8, 30.4, 29.7, 29.6, 29.3, 26.2, 26.1, 24.4, 22.7 (21 C, some
signals were overlapped, 2 OCH2(CH2)10CH3, OCH2CH2CH2N(CH3)3), 14.0, 14.0 (2 C, 2
OCH2CH2(CH2)9CH3). ESI-MS: m/z = 616.8, in agreement with the calculated mass for [M]+ =
+
C36H74NO6 .
3.2.2 Synthesis of Cationic Glycolipids 4 (Scheme 2)
3’-aminopropyl-β-D-glucopyranoside (19)
The mixture of compound 13 (3.9 g, 14.8 mmol), THF (50.0 mL), H2O (5.0 mL), and PPh3
(7.8 g, 29.6 mmol) was refluxed at 75oC until TLC (petroleum ether : ethyl acetate = 2 : 1) showed
the starting material disappeared. The reaction mixture was evaporated to dryness, H2O (50.0 mL)
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was dropped to it, and a white solid would be given, then the mixture was filtered andDaOI:y1e0l.l1o03w9/C5OB02107C
syrup 19 (3.0, 85.4%) would be given when the filtrate was evaporated to dryness.
3’-[(N,N-di-dodecylamino]-propyl-β-D-glucopyranoside (20a)
The mixture of compound 18 (1.2 g, 5.1 mmol), 1-bromododecane (5.1 g, 20.4 mmol),
anhydrous K2CO3 (1.4 g, 10.2 mmol), CH3OH (20.0 mL), CH3CH2OH (20.0 mL) was refluxed at
75oC until TLC (methanol) showed the starting material disappeared. The mixture was diluted
with DCM (30.0 mL), and washed with water for two times. The organic layer was dried by
anhydrous Na2SO4 and concentrated to dryness. The residue was purified by silica gel column
chromatography with EtOAc : methanol = 4 : 1 as the eluent to give compound 20a (1.2 g, 41.3 %)
as a syrup. 1H NMR (500 MHz, CDCl3): δ (ppm): 4.30 (d, 1 H, J1,2 = 7.5 Hz, H-1), 3.95-3.85 (m, 1
H, OCHHCH2CH2N(CH2CH2(C9H18)CH3)2), 3.88-3.70 (m, 2 H, H-6), 3.64-3.50 (m, 3 H,
OCHHCH2 CH2N(CH2CH2(C9H18)CH3)2), H-3, H-4), 3.39 (dd, 1 H, J2,1 = 7.5 Hz, J2,3 = 3.5 Hz,
H-2), 3.30-3.28 (m, 1 H, H-5), 2.58-2.50 (m, 2 H, OCH2CH2CH2N(CH2CH2(C9H18)CH3)2), 2.44 (t,
J = 6.5 Hz, 4 H, N(CH2CH2(C9H18)CH3)2), 1.89-1.75 (m, 2 H, OCH2CH2CH2N(CH2CH2
(C9H18)CH3)2), 1.49-1.40 (m, 4 H, N(CH2CH2(C9H18)CH3)2), 1.37-1.21 (m, 36 H, N(CH2CH2
(C9H18)CH3)2), 0.89 (t, 6 H, J = 6.5 Hz, N(CH2CH2(C9H18)CH3)2); 13C NMR (125 MHz, CDCl3):
δ (ppm) 103.1 (1 C, C-1), 76.5 (1 C, C-3), 75.8 (1 C, C-5), 73.5 (1 C, C-2), 69.6 (1 C, C-4), 68.7
(1 C, OCH2CH2CH2N(CH2CH2(C9H18)CH3)2), 61.4 (1 C, C-6), 53.4, 53.4 (2 C, N(CH2CH2(C9H18)
CH3)2), 50.7 (1 C, OCH2CH2CH2N(CH2CH2(C9H18)CH3)2), 31.9, 29.8, 29.7, 29.6, 29.3, 27.7, 27.2,
25.7, 22.6 (21 C, some signals were overlapped, N(CH2(C10H20)CH3)2), OCH2CH2CH2N
(CH2CH2(C9H18)CH3)2), 14.1, 14.1 (2 C, N(CH2(C10H20)CH3)2).
3’-[(N,N-di-dodecyl-N-methyl)aminonium iodine]-propyl-β-D-glucopyranoside (21a, lipid 4)
The mixture of compound 20a (250.0 mg , 0.44 mmol) and iodomethane (250.0 mg, 1.76
mmol, 109.0 µL), THF (5.0 ml) was stirring at the room temperature until TLC (ethyl acetate :
methanol = 5 : 1) showed the starting material disappeared. The reaction mixture was evaporated
to dryness and a solid was precipitated when the acetone (10.0 mL) was dropped to the syrup. The
mixture was filtered, and the filter cake was washed with acetone (5.0 mL × 3) and dried by
vacuum to give white solid lipid 4 (270.0 mg, 86.8%). 1H NMR (500 MHz, CDCl3): δ (ppm): 5.09
(s, 1 H, OH), 4.88 (s, 2 H, 2 OH), 4.46 (d, 1 H, J1,2 = 7.5 Hz, H-1), 4.10-4.00 (m, 2 H, OH,
OCHHCH2CH2N(CH3)(C12H25)2), 3.86-3.75 (m, 3 H, H-6, OCHHCH2CH2N(CH3)(C12H25)2),
3.74-3.50 (m, 4 H, H-3, H-4, OCH2CH2CH2N(CH3) (C12H25)2), 3.44-3.30 (m, 6 H, H-2, H-5,
(CH3)N(CH2CH2(C9H18)CH3)2), 3.21 (s, 3 H, (CH3)N(CH2CH2(C9H18)CH3)2), 2.22-2.14 (m, 2 H,
OCH2CH2CH2N(CH3)(C12H25)2), 1.75-1.67 (m, 2 H, (CH3)N(CH2CH2(C9H18)CH3)2,) 1.40-1.20
(m, 36 H, (CH3)N(CH2CH2(C9H18)CH3)2), 0.86 (t, 6 H, J = 6.5 Hz, (CH3)N(CH2CH2
(C9H18)CH3)2); 13C NMR (125 MHz, CDCl3): δ (ppm) 102.7 (1 C, C-1), 76.2 (1 C, C-3), 75.9 (1 C,
C-5), 73.2 (1 C, C-2), 69.8 (1 C, C-4), 66.2 (1 C, OCH2CH2CH2N(CH3)(C12H25)2), 61.2, 61.2,
61.2 (3 C, OCH2CH2CH2N(CH3)(CH2CH2(C9H18)CH3)2), 60.8 (1 C, C-6), 49.4 (1 C,
(CH3)N(CH2CH2(C9H18)CH3)2), 31.9, 29.6, 29.5, 29.4, 29.3, 29.1, 26.3, 23.5, 22.6 (21 C, some
signals were overlapped, (CH3)N(CH2(C10H20)CH3)2), OCH2CH2CH2N(CH3)(C12H25)2), 14.1, 14.1
(2 C, (CH3)N(CH2(C10H20)CH3)2). ESI-MS: m/z = 588.7, in agreement with the calculated mass
for [M]+ = C34H70NO6 .
+
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3.3 Preparation of liposomes and DNA-Lipid Complexes. Each glycolipid was disappeared into
sterile double distilled water. The mixture was ultrasonaticed at 37oC for 30 min in a closed vial to
give a clarity solution, and filtered with syringe filter (0.45µm) to form the solution of liposome.
The concentration of liposome was 0.5 mmol/L for lipids 1-3, 0.25 mmol/L for lipids 4-6.
Lipoplexs with different N/P ratio were prepared by mixed the plasmid DNA (1µg) with various
amounts of liposomal solution.
3.4 Gel Retardation. To examine the complexation of DNA with cationic lipid suspensions at
different lipid-DNA ratios, we prepared lipid-DNA complexes at different charge ratios varying
from 0.3:1 to 3:1(lipid/DNA) to measure the DNA binding ability. After 30 min of incubation,
these complexes were electrophoretically run on a 1.0% agarose gel containing (0.2 μg/mL)
ethidium bromide for DNA staining. The gel was placed in a horizontal electrophoresis apparatus
(DYY-6C, Beijing Liuyi Biotechnology Co. Ltd.) containing ×1 Tris acetate EDTA (TAE) buffer
solution (prepared in our lab as ×5 stock solution), exposed to an electric field (320 V) for 20 min,
and then visualized by Gel image system (Tanon 2500, Shanghai Tianneng science and
Technology Co. Ltd).
3.5 Lipoplex and lipids particle sizes and zeta potentials. The average particle size and charge
of the different lipids and various N/P ratios were determined by quasielastic laser light scattering
with a Malvern Zetasizer (Nano-ZS, Malvern Instruments). Each sample was analyzed in triplicate,
and the results were reported as the means ± standard deviation. The lipoplex particle solution was
first prepared by mixing liposomes and pEGFP (1 μg/μL) under diverse N/P charge ratios in 1 mL
deionized water.
3.6 Morphology study by atomic force microscopy. The samples for AFM measurements were
prepared by mixing lipids and 0.8 μg pEGFP in 100 μL of distilled water under the best N/P ratios
and incubated for 20 min, and the solution mixture were dropped onto a mica slice, and then the
solvent was completely evaporated in the air under the room temperature prior to AFM
measurements. Morphologies of the lipids/pEGFP lipoplexes were characterized at room
temperature on a NanoScope IV atomic force microscopy (AFM, Veeco Instruments, Co. Ltd.)
with a tapping mode.
3.7 Cell Culture. Cells (Hek293) were cultured in Dulbecco,s Modified Eagle,s Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin,
10,000 U mL-1) in T55 culture dish and were incubated at 37oC in a humidified atmosphere
containing 5% CO2. Cells were regularly passaged by trypsinization with 0.1% trypsin (EDTA
0.02%, dextrose 0.05%, and trypsin 0.1%) in PBS (pH = 7.2).
3.8 Transfection of Cells. Transfection of the cells was done essentially as described earlier. In
order to obtain about 80% confluent cultures at the time of transfection, 24-well plates were
seeded with 100,000 cell/well in 500 μL mediated 24 h before transfection. For the preparation of
lipoplexes applied to cells, various amounts of liposomes and DNA were serially diluted
separately in serum-free DMEM culture medium; then, the DNA solutions were added into
liposome solutions drop by drop and vortexed gently, after which the mixtures were incubated at
room temperature for about 30 min to obtain lipoplexes. The DNA was used at a concentration of
0.8 μg/well unless otherwise noted. After 30 min of complexation, old cell culture medium was
removed from the wells, cells were washed once with PBS, and the above lipoplexes was added to
each well. The plates were then incubated for 4 h at 37oC in a humidified atmosphere containing 5%
CO2. At the end of incubation period, medium was removed and 500 μL of fresh DMEM culture
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medium containing 10% FBS was added to each well. Plates were further incubated for a period
of 24 h before checking the reporter gene expression. For fluorescent microscopy assays, cells
were transfected by complexes containing pEGFP-C3. After 24 h incubation, the microscopy
images were obtained at the magnification of 20 and recorded using IpWin51C image analysis
system. Control transfection was performed in each case using a commercially available
transfection reagent Lipofectamine 2000 based on the standard conditions specified by the
manufacture.
Cell count of transfection efficiency [49]. After the old cell culture medium was removed from
the wells and washed with PBS, 50 uL of parenzyme was introduced to the 24-well plates. When
the pates were incubation for 2-3 min at 37oC in a humidified atmosphere containing 5% CO2, 200
μL of DMEM was added and the well-distributed suspension was afforded by scattering the cells.
Then, 100 μL of cell suspension was removed to the centrifugal tube and used to cell count by
diluting with another 100 μL of DMEM. Afterwards, 10 μL of the diluted suspension was
introduced to serum counting plate, white and fluorescence images were given by fluorescent
microscopy in the same position. Three samples of per well were taken out, five white and
fluorescence images were afforded by fluorescent microscopy for each sample. Finally, count cells
in the white and fluorescence images, and then the y% transfection efficiency of glycolipids were
calculated as (cell numbers in the fluorescence images / cell numbers in the white images) × 100.
In the same way, x% transfection efficiency of lipo2000 (as a positive control) was obtained. Then
the relative transfection efficiency of glycolipid to lipo2000 was calculated according to the
푦%
formula:
× 100.
푥%
3.9 Cytotoxicity. Toxicity of each cationic lipid formulation toward Hek293 cells was determined
using 3-(4,5-dimethylthiaz-ole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay as
described earlier [51-52]. In order to obtain accurate results, cytotoxicity levels of the lipid
formulations optimal for transfection experiments were found out under conditions exactly similar
to transfection conditions. About 30 000 cells/well were plated in 96 well plates. After 24 h,
various N/P ratios of lipids were added to the cells in the absence of serum. After 4 h of incubation,
20 µL of MTT solution was added. Blue Formosan crystals were seen when checked under
microscope. Media was removed and 150 µL of DMSO was added per well. The absorbance was
measured using a microtiter plate reader. The % viability was then calculated as [{A490 (treated
cells) - background]/[A490 (untreated cells) - background}] × 100.
4. Conclusions
In this study, two series of glucose-based cationic lipids were synthesized, and their utility in
a pEGFP-encapsulating liposome mediated gene delivery system were investigated. The size,
ζ-potential, toxicity and the capacity of DNA-banding of glycolipids were also detected. The
transfect efficiency of glycolipids with hydrophobic tails linked to the positively charged nitrogen
atoms were higher than that of glycolipids with hydrophobic tails linked to the glucose cyclic
directly. Most importantly, among of six glycolipids, lipid 6 with the hexadecyl as hydrophobic
tail linked to the positively charged nitrogen atoms was observed the highest gene expression
efficiency and lower cytotoxicity in HEK293 cells. We are currently focusing our efforts on
performing a detailed structure/activity relationship analysis to gain a better insight and
understanding of these glucose-based cationic lipids and will apply it to disease models based on
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siRNA therapeutics.
Acknowledgements
We are grateful for financial support from Nation Nature Sciences Foundation of China
(General Program: 21272064) and Science & Technology Department of Changsha City
(K1205221-31). We would like to thank Dr. Ying Zeng (College of Chemistry and Chemical
Engineering, Hunan Normal University) and Dr. Zhonghua Liu (School of Life Sciences, Hunan
Normal University) for their expert help on cell culture and transfect efficiency assay.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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