Preparation, physicochemical properties, and transfection activities of tartaric acid-based cationic lipids as effective nonviral gene delivery vectors

Article abstract of DOI:10.1248/bpb.b16-00007

In this work two novel cationic lipids using natural tartaric acid as linking backbone were synthesized. These cationic lipids were simply constructed by tartaric acid backbone using head group 6-aminocaproic acid and saturated hydrocarbon chains dodecanol (T-C12-AH) or hexadecanol (T-C16-AH). The physicochemical properties, gel electrophoresis, transfection activities, and cytotoxicity of cationic liposomes were tested. The optimum formulation for T-C12-AH and T-C16-AH was at cationic lipid/dioleoylphosphatidylethanolamine (DOPE) molar ratio of 1:0.5 and 1:2, respectively, and N/P charge molar ratio of 1:1 and 1:1, respectively. Under optimized conditions, T-C12-AH and T-C16-AH showed effective gene transfection capabilities, superior or comparable to that of commercially available transfecting reagent 3β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and N-[2,3-dioleoyloxypropyl]-N,N,N-trimethylammonium chloride (DOTAP). The results demonstrated that the two novel tartaric acid-based cationic lipids exhibited low toxicity and efficient transfection performance, offering an excellent prospect as nonviral vectors for gene delivery.

Full text of DOI:10.1248/bpb.b16-00007

1112
Biol. Pharm. Bull. 39, 1112–1120 (2016)
Vol. 39, No. 7
Regular Article
Preparation, Physicochemical Properties, and Transfection Activities of
Tartaric Acid-Based Cationic Lipids as Effective Nonviral Gene Delivery
Vectors
Ning Wan,a,# Yi-Yang Jia,a,# Yi-Lin Hou,b Xi-Xi Ma,a Yong-Sheng He,a Chen Li,a
,a
Si-Yuan Zhou,a and Bang-Le Zhang*
a Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University; Xi’an 710032, China: and
b Innovative Experimental College, Northwest A&F University; Yangling 712100, China.
Received January 5, 2016; accepted March 23, 2016; advance publication released online April 26, 2016
In this work two novel cationic lipids using natural tartaric acid as linking backbone were synthesized.
These cationic lipids were simply constructed by tartaric acid backbone using head group 6-aminocaproic
acid and saturated hydrocarbon chains dodecanol (T-C12-AH) or hexadecanol (T-C16-AH). The physico-
chemical properties, gel electrophoresis, transfection activities, and cytotoxicity of cationic liposomes were
tested. The optimum formulation for T-C12-AH and T-C16-AH was at cationic lipid/dioleoylphosphatidyl-
ethanolamine (DOPE) molar ratio of 1:0.5 and 1:2, respectively, and N/P charge molar ratio of 1:1 and
1:1, respectively. Under optimized conditions, T-C12-AH and T-C16-AH showed effective gene transfection
capabilities, superior or comparable to that of commercially available transfecting reagent 3β-[N-(Nꢀ,Nꢀ-
dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and N-[2,3-dioleoyloxypropyl]-N,N,N-trimethylam-
monium chloride (DOTAP). The results demonstrated that the two novel tartaric acid-based cationic lipids
exhibited low toxicity and efficient transfection performance, offering an excellent prospect as nonviral vec-
tors for gene delivery.
Key words nonviral vector; cationic lipid; tartaric acid; gene transfection
Nowadays, gene therapy is considered to be a powerful ap- which can give a favorable gene delivery efficiency and low
proach in curing a multitude of diseases, especially in devel- cytotoxicity.
oping strategies for the prevention and treatment of many dis-
Natural tartaric acid is inexpensive and readily available
eases, such as cancer, AIDS and so on.1,2) The vectors used for and widely used in drinks and other foods.40) Tartaric acid, a
gene transfection play the key role in successful gene therapy, multi-functional molecule, has two hydroxyl and two carboxyl
which are roughly divided into viral and nonviral ones.3–5) groups as reactive sites, which is suitable as the backbone in
Viral vectors showed high tranfection efficiency for gene de- the design of cationic lipids through easily modified with dif-
livery,6,7) but the huge disadvantages of viral vectors limited ferent head group and alkyl hydrophobic tail.
their clinical applications, such as immunogenic responses,
high cost of production and limitation of the exogenous DNA to design and synthesize the cationic lipids for gene delivery.
size.8–13)
The cationic lipids were simply constructed by tartaric acid
In this paper, natural tartaric acid was used as a backbone
Since the application of viral vectors are limited, nonviral backbone using polar head group 6-aminocaproic acid21) and
vectors including cationic lipids, cationic polymers have been saturated hydrocarbon chains dodecanol (T-C12-AH) or hexa-
drawing more and more attention nowadays.14–24) Among the decanol (T-C16-AH) (Fig. 1). The liposome formulations for
existing nonviral vectors, the cationic liposome, represent- gene delivery were prepared and optimized by introducing
ing an attractive, alternative approach for gene delivery has helper lipid dioleoylphosphatidylethanolamine (DOPE) at vari-
a broad varity of advantages, such as biodegradability, easy ous cationic lipid/DOPE ratios and cationic lipid/DNA ratios.
preparation, good repeatability and potential clinical applica- The cytotoxicity of the cationic liposomes was also evaluated.
tions.25–29)
Cationic lipids generally consist of polar head group
and hydrophobic tails connected through the backbone
(Fig. 1), while most of the backbone in earlier cationic
MATERIALS AND METHODS
Materials Natural tartaric acid, dodecanol, hexadecanol,
lipids was classified into glycerol-type just as N-[2- 6-aminocapraic acid (AH), di-tert-butyl dicarbonate ((Boc)2O),
[(1,5,10,14-tetraazatetradecane-1-yl)carbonylamino]ethyl]-N,N- dicyclohexylcarbodiimide (DCC), trifluoroacetic acid (TFA)
dimethyl-2,3-bis(oleoyloxy)-1-propanaminium (DOSPA) and and 4-dimethylaminopryidine (DMAP) were purchased from
N-[2,3-dioleoyloxypropyl]-N,N,N-trimethylammonium chlo- Sun Chemical Technology (Shanghai) Co., Ltd. All starting
ride (DOTAP)30,31) and cholesterol-type, such as 3β-[N-(N′,N′- materials and reagents were used without further purifica-
dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).32) Re- tion. Silica gel (300–400 mesh) and potassium bromide (KBr,
cently, phosphonate,33) bile acid,34) amino acids,35,36) pep- spectroscopic grade) were purchased from Xi’an Ke Hao Bio-
tides,37) pentaerythritol38) and carbohydrate39) were also suc- logical Engineering Co., Ltd. (China). DOPE was bought from
cessfully used as backbones in the design of cationic lipids, Fluka (Buchs, Switzerland). DC-Chol was bought from Sigma
(St. Louis, MO, U.S.A.). pEGFP-N1 encoding the enhanced
# These authors contributed equally to this work.
green fluorescence protein (GFP) was purchased from Shang-
*To whom correspondence should be addressed. e-mail: blezhang@fmmu.edu.cn
© 2016 The Pharmaceutical Society of Japan

Vol. 39, No. 7 (2016)
Biol. Pharm. Bull.
1113
Fig. 1. Cationic Lipids with Different Backbone Developed for Gene Delivery
hai GenePharma Co., Ltd. (Shanghai, China). Fetal bovine tracted with 150mL CH2Cl2 and washed with deionized water
serum (FBS), opti-minimal essential media (MEM) and Dul- for 3 times. Organic solvent was evaporated under reduced
becco’s modified Eagle’s medium (DMEM) were bought from presure and the product was dried in a vaccum oven (45°C)
Gibco (Carlsbad, CA, U.S.A.).
to afford yellowish thick liquid, 2.13g, yield: 61.6%. MS m/z
Cells HEK 293T cells and HeLa cells were bought from electronspray ionization (ESI)+: 232 (M)+. IR (KBr) cm−1:
the Culture Collection of the Chinese Academy of Science 3348, 2978, 2939, 2719, 2646, 1670, 1651, 1504, 1273, 1173,
(Shanghai, China). The cells were grown in DMEM with 10% 864, 779. 1H-NMR (CDCl3) δ: 1.38–1.34 (2H, t, J=7.8Hz),
FBS at 37°C in 5% CO2, penicillin at 100UmL−1 and strepto- 1.43 (9H, s), 1.53–1.47 (2H, m), 1.68–1.61 (2H, m), 2.37–2.33
mycin at 100µgmL−1.
(2H, t, J=7.4Hz), 3.12–3.09 (2H, t, J=6.1Hz), 4.56–4.55 (1H,
Characterization of the Synthesized Compounds TLC d, J=0.6 Hz).
was performed to test the reactions. Purification was carried
Synthesis of Compound 2
out by silica gel column chromatography. IR spectra were
Compound 2 was prepared according to the previous
recorded using a Fourier transform (FT)-IR spectrometer. All study.41) In brief, tartaric acid (1, 4.90g, 32.65mmol), dodeca-
samples to be tested were ground and compressed with KBr nol (13.38g, 71.83mmol) and concentrated hydrochloric acid
1
into a thin disk under hydraulic press. H-NMR spectra were (0.7mL) were added into a round-bottom flask. The reaction
recorded at 400MHz. Mass spectra were detected by Quattro solution was heated under stirring for 36h at 120°C. The mix-
Premier Micromass.
ture was then cooled to room temperature. The obtained white
Synthesis of the Cationic Lipids T-C12-AH and T- solid was rinsed by 1mol/L sodium hydrate solution (3×6mL)
C16-AH
Synthesis of Boc-AH
and a small amount of water. Then the resulting solid was
recrystallized for two times from ethanol to give compound 2,
6-Aminocapraic acid (2.00g, 15.25mmol) was dissolved 11.40g, white solid, yield: 71.7%, mp: 65–66°C [62–64°C (41)].
in 40mL NaOH solution (0.62mol/L). Then (Boc)2O (3.66g, MS m/z ESI+: 488 (M)+. IR (KBr) cm−1: 3445, 2916, 2847,
1
16.77mmol) in 22mL tetrahydrofuran (THF) was added drop- 1720, 1635, 1277, 1103, 1068, 876, 748. H-NMR (CDCl3) δ:
wise into the flask at 0°C. The mixture was stirring for 30min 0.89–0.86 (6H, t, J=5.1Hz), 1.26 (36H, m), 1.70–1.65 (4H, m),
at 0°C and then reacted at room temperature for 24h. A rotary 4.27–4.23 (4H, m), 4.52 (1H, s).
evaporator was used to remove the THF. Then 100mL Et2O
was applied to extract the unreacted (Boc)2O. One mol/liter
Synthesis of Compound 3
Tartaric acid (1, 4.90g, 32.65mmol), hexadecanol (17.41g,
HCl was added dropwise into the aqueous phase until the 71.83mmol) and concentrated hydrochloric acid (0.7mL) were
pH was approximately 3. The aqueous phase was then ex- added into the 100mL round-bottom flask. The reaction solu-

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Biol. Pharm. Bull.
Vol. 39, No. 7 (2016)
tion was heated under stirring for 24h at 120°C. Then the white solid, yield: 77.3%, mp: 51–53°C. MS m/z ESI+:
mixture was cooled to room temperature, and the obtained 826 (M−CF3COOH−CF3COO−)+, 413 (M−2CF3COO−)2+.
white solid was rinsed by 1mol/L sodium hydrate solution IR (KBr) cm−1: 3418, 2916, 850, 1751, 1682, 1273, 1203,
1
(3×6mL) and a small amount of water, recrystallized from 1134, 1076, 841, 741. H-NMR (CDCl3) δ: 0.89–0.86 (6H, t,
ethanol two times to give compound 3, 14.31g, white solid, J=6.8Hz), 1.25 (s, 52H), 1.48–1.45 (4H, m), 1.78–1.61 (12H,
yield: 73.2%, mp: 78–79°C. IR (KBr) cm−1: 3479, 2916, 2847, m), 2.96–2.94 (4H, t, J=7.2Hz), 2.43–2.39 (4H, m), 4.16–4.13
1759, 1716, 1288, 1192, 1134, 1068, 879, 717. 1H-NMR (CDCl3) (4H, t, J=6.6Hz), 5.67 (2H, s), 7.87 (6H, s).
δ: 0.88 (6H, s), 1.25 (52H, s), 1.70–1.67 (4H, t, J=7.2Hz),
4.28–4.24 (4H, m), 4.52 (2H, s).
Preparation of Liposomes and Lipoplexes Liposomes
were prepared through thin-film hydration method. DOPE and
lipids were taken in desired molar ratios and dissolved in ap-
Synthesis of Compound 4
DCC (0.76g, 3.70mmol), DMAP (4mg, 34.4µmol) and propriate amount of chloroform, solvent was slowly removed
compound 2 (0.30g, 0.62mmol) were added to a solution of under vacuum. The resulting film was placed in vacuum oven
Boc-AH (0.57g, 2.47mmol) in dry CH2Cl2 (10mL) at 0°C (45°C) overnight and then hydrated in deionized water to the
stirring for 1h. Then the mixture was stirring for another 5h final cationic lipid concentration of 1mM. Hydration process
at room temperature. After filtering, the filtration was evapo- continued at 4°C for 12h. The hydration solution was vortex-
rated under reduced presure, then the resulting residue was mixed for about 5min and sonicated for another 15min. Li-
further purified by column chromatography (petroleum ether posomes were then extruded through filter with porosity of
(PE):EtOAc, 5:1) to give compound 4, 0.32g, white solid, 0.45 µm and 0.2µm for six times respectively and stored at
yield: 85.3%, mp: 62–63°C. MS m/z ESI+: 914 (M)+. IR (KBr) 4°C. Lipoplexes were prepared as followed. The cationic lipo-
cm−1: 3375, 2924, 2850, 1770, 1751, 1686, 1516, 1250, 1157, somes diluted with an appropriate amount of opti-MEM were
1
1041, 999, 868, 729. H-NMR (CDCl3) δ: 0.89–0.86 (6H, t, mixed with pEGFP-N1 diluted with dd H2O. The mixture was
J=6.4Hz), 1.25–1.22 (36H, m), 1.35–1.33 (4H, m), 1.43 (18H, then gently vortexed and incubated for 30min at room temper-
m), 1.67–1.60 (12H, m), 2.45–2.31 (4H, m), 3.11–3.10 (4H, d, ature to form lipoplexes (cationic liposome/DNA complexes).
J=2.4Hz), 4.16–4.13 (4H, t, J=5.4Hz), 4.62 (2H, s), 5.70 (2H, The lipoplexes were then diluted with an appropriate amount
s).
of opti-MEM for analyzing the gene transferring efficiency.
Synthesis of Compound 5
Cationic liposome/DNA lipoplexes were prepared at a DNA
DCC (0.84g, 4.13mmol), DMAP (4mg, 34.4µmol) and concentration of 25µg/mL for the measurement of particle
compound 3 (0.41g, 0.69mmol) were added to a solution of size and zeta potential. The lipoplexes formed by the lipo-
Boc-AH (0.64g, 2.75mmol) in dry CHCl3 (10mL) at 0°C somes of DOTAP or DC-Chol/DOPE (1:1, molar ratio) with
and stirred for 1h. Then the mixture was stirred at room plasmid DNA (pDNA) at the optimal N/P ratio of 1:1 were
temperature for another 5h. After filtering, the filtration was used as the positive control groups.
evaporated under reduced presure, then the resulting residue
Measurement of Size Distribution and Zeta-Potential of
was further purified by column chromatography (PE:EtOAc, Cationic Liposomes and Lipoplexes The particle size and
6:1) to give compound 5, 0.64g, White solid, yield: 91.2%, zeta potential of cationic liposomes and cationic liposome/
mp: 45–46°C. MS m/z ESI+: 1026 (M)+. IR (KBr) cm−1: 3402, DNA lipoplexes were determined with the Delsa™ Nano C
2924, 2854, 1751, 1716, 1701, 1520, 1273, 1250, 1173, 1150, Particle Analyzer (Beckman Coulter) by the dynamic light
1
868, 721. H-NMR (CDCl3) δ: 0.89–0.86 (6H, t, J=6.4Hz), scanning method, and were determined for 3 times. Data were
1.25 (52H, m), 1.40–1.34 (4H, m), 1.51–1.43 (18H, m), analyzed using the ELS-Z software package supplied by the
1.68–1.60 (12H, m), 2.45–2.38 (4H, m), 3.13–3.09 (4H, m), manufacturer.
4.16–4.11 (4H, m), 4.61 (2H, s), 5.70 (2H, s).
Gel Retardation Assay The gel electrophoresis assay was
Synthesis of T-C12-AH
carried out to evaluate the electrostatic interactions and opti-
Trifluoroacetic acid (1mL) was added to the solution of mize the lipid/DNA ratios between cationic lipids and DNA.
compound 4 (0.38g, 0.42mmol) in CH2Cl2 (10mL) at 0°C In brief, 0.8µg pDNA was mixed with liposomes at different
for 30min, and reacted for another 4h at room tempera- N/P ratios. The ethidium bromide intercalating agent was used
ture. Then CH2Cl2 was evaporated under reduced presure, as staining reagent. Electrophoresis was performed in 0.5×
and the residue was purified by column chromatogra- Tris borate ethylenediaminetetraacetic acid (TBE) running
phy (CH2Cl2 :MeOH:H2O, 80:10:1) to obtain T-C12-AH buffer at 100V for 50min. The gel images were taken by a
0.34g, light yellow oil, yield: 80.0%. MS m/z ESI+: 714 UV light illuminator.
(M−CF3COOH−CF3COO−)+, 358 (M−2CF3COO−)2+. IR
Transfection Activity The DNA delivery efficiency of
(KBr) cm−1: 3425, 3070, 2933, 2854, 1751, 1678, 1277, 1204, synthesized cationic lipids was determined in 293T cells using
1
1138, 1072, 837, 798. H-NMR (CDCl3) δ: 0.89–0.86 (6H, t, GFP as a reporter gene. The transfection activities of the
J=6.8Hz), 1.27–1.22 (36H, m), 1.48–1.43 (4H, m), 1.70–1.61 cationic liposomes were tested by flow cytometry and fluores-
(12H, m), 2.42–2.38 (4H, m), 2.96–2.92 (4H, t, J=7.2Hz), cence microscopy.
4.16–4.13 (4H, t, J=6.4Hz), 5.66 (2H, s), 7.92–7.90 (6H, m).
Synthesis of T-C16-AH
As for flow cytometry assay, 293T cells or HeLa cells were
seeded on 6-well plates at 200000cells/well in DMEM con-
Trifluoroacetic acid (1mL) was added to the solution of taining 10% FBS for 24h before transfection. The cells were
compound 5 (0.54g, 0.53mmol) in CHCl3 (10mL) at 0°C rinsed with DMEM (1mL) and another 800µL serum free
for 30min, and reacted for another 7h at room tempera-
ture. Then CH2Cl2 was evaporated under reduced presure,
and the residue was purified by column chromatography
(CH2Cl2 :MeOH:H2O, 80:12:1) to obtain T-C16-AH 0.43g,
DMEM was added. Two hundred microliters of the lipoplex
formulations (containing 2.5µg DNA) in opti-MEM were
added to cells, and incubated for 8h at 37°C. Transfection
media was then replaced by 2mL of DMEM containing 10%

Vol. 39, No. 7 (2016)
Biol. Pharm. Bull.
1115
Reagents and Conditions: (a) dodecanol or hexadecanol, concentrated HCl, 120°C; (b) Boc-AH, N,N′-dicyclohexylcarbodiimide (DCC)/4-dimethylamiopryidine
(DMAP), CHCl3 for 2, CH2Cl2 for 3; (c) TFA, CH2Cl2.
Fig. 2. Synthetic Route of T-C12-AH and T-C16-AH
FBS, and the cells were incubated for another 40h. The cells Table 1. Particle Sizes and Zeta Potentials of Cationic Liposomes at
Lipid/DOPE Molar Ratio of 1:1
were collected for flow cytometry using a Becton and Dick-
inson flow cytometer. Both the mean fluorescence intensity
(MFI) and the percent of positive transfected cells had been
recorded for transfection activity evaluation.
When tested by fluorescence microscopy, 293T cells were
plated at 24-well plates at 50000cells/well, and incubated
for 24h. The desired lipid formulation of different N/P ratios
Size
(nm)
Polydispersity
(PDI)
Zeta potential
(mV)
Cationic lipid
T-C12-AH
T-C16-AH
78.2 0.4
83.7 0.5
0.254 0.002
0.236 0.001
58.6 3.0
58.4 2.3
and DNA (0.8µg per well) were complexed in opti-MEM fied to the carboxyl groups of tartaric acid to prepare cationic
and incubated for 30min at room temperature. Next, original lipid in three steps with high yield and the effect of hydropho-
cell cultures was discarded, and cells were rinsed once with bic chain length on the transfection efficiency was examined.
DMEM. Then, the lipoplexes mentioned above were added to The yield of compounds 2 and 3 after recrystallization was
the cells. After incubation at 37°C for 8h, transfection media 71.7 and 73.2%, respectively. The protected 6-aminocaproic
were removed, and replaced by 500µL DMEM containing acid derivative Boc-AH21) was reacted with the compounds
10% FBS. The transfection was stopped after incubation for 2 or 3 via an amide linkage to synthesize compounds 4 or
another 40h. Fluorescence microscopy was applied to exam- 5. It was worth noting that compound 2 had better solubility
ine GFP expression.
in CHCl3 than CH2Cl2, so we chose CHCl3 as the solvent for
Cytotoxicity Assay The cytotoxicity of optimized for- compound 2. After deprotection with trifluoroacetic acid, two
mulation of cationic liposomes was also investigated with novel tartaric acid based cationic lipids were obtained. The
1
293T cells. Briefly, cells were seeded on 96-well plates at structure of all compounds was identified by H-NMR, IR and
20000cells/well and incubated for 24h under 5% CO2 at 37°C MS spectra and was consistent with the targeted compounds.
and cultured with 200µL of DMEM containing 10% FBS. The trifluoroacetate was confirmed by 1H-NMR spectra of
Then 200µL of DMEM containing different concentration of compounds T-C12-AH and T-C16-AH, in which there were
the liposomes was added to replace the cell culture medium. typical proton signals of –NH2+ at 7.91 and 7.87ppm, respec-
Tewnty microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen- tively.
yltetrazolium bromide (MTT) solution was added to the cells
Preparation and Characterization of Cationic Lipo-
24h post exposure to the different concentration of cationic somes Cationic lipids were often mixed with a neutral co-
liposomes. The cells were incubated for further 4h. Then cell lipid dioleoylphosphatidylethanolamine (DOPE) to formulate
medium was discarded, and each well was treated with 150µL into cationic liposomes.42–47) The results of the particle size,
of dimethyl sulfoxide (DMSO) to fully dissolve the reduced size distribution and zeta potential of the cationic liposomes
crystal violet. Finally a microtiter plate reader was used to were shown in Table 1. The cationic formulation of two cat-
determine the absorbance of the solution at 490nm.
ionic lipids at lipid/DOPE ratio of 1:1 exhibited rather small
hydrodynamic diameters 78.2nm for T-C12-AH and 83.7nm
for T-C16-AH. The positive potential was 58.6 and 58.4mV,
respectively. These results indicated that the hydrophobic do-
RESULTS AND DISCUSSION
Synthesis of the Cationic Lipids T-C12-AH and T-C16- main of cationic lipids exerted little influence on the physical
AH The cationic lipid materials T-C12-AH and T-C16-AH properties of liposomes.
were constructed as shown in Fig. 2. The double chain hydro-
Agarose Gel Retardation Assay Agarose gel retarda-
carbons are normally ranging from 12 to 18 carbon units in tion assay was carried out to evaluate the binding interactions
length,28) so dodecanol and hexadecanol were used and esteri- between cationic liposomes and pDNA at different N/P ratios.

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Vol. 39, No. 7 (2016)
Fig. 3. Agarose Gel Retardation of DNA Lipoplexes of T-C12-AH (A) and T-C16-AH (B) at Various N/P Ratios
Fig. 4. Effects of DOPE on the Transfection Activity of T-C12-AH (A) and T-C16-AH (B) in 293T Cells
The data of the percent of positive transfected cells (GFP Cell%) and mean fluorescence intensity (MFI) obtained from flow cytometry analysis. Data are shown as the
mean S.D. (n=3). *p<0.05, **p<0.01, compared to the percent of positive transfected cells at a lipid/DOPE ratio of 1:1. # p<0.05, ## p<0.01, compared to the MFI at a
lipid/DOPE ratio of 1:1.
Stable cationic liposome/DNA complexes were not able to the increase of the ratio of DOPE to 3. At lipid/DOPE ratio of
penetrate into the agarose gel, on contrast, free DNA or DNA 1:2, the MFI of T-C16-AH liposome was highest among oth-
not fully combined to cationic liposomes could penetrate into ers. While for lipid T-C12-AH, the transfection activities re-
the agarose gel. As shown in Fig. 3, between N/P ratio of 1:4 duced with the increase of DOPE ratio and the optimal lipid/
and 5:1, compared with free DNA, the free mobile DNA dis- DOPE ratio was 1:0.5. Although the MFI of lipid T-C12-AH
appeared at N/P ratio of 1:1 (cationic lipid/DNA molar ratio at lipid/DOPE ratio of 1:1 was close to that of at lipid/DOPE
of 0.5:1) for both cationic lipids T-C12-AH and T-C16-AH. ratio of 1:0.5, the percent of positive transfected cells was
In addition, when N/P ratio was over 1:1, DNA was entirely lower. The images of GFP expression observed by a fluores-
compacted and protected in the cationic liposomes, the fluo- cence microscope were in line with the aboved results (Fig.
rescent in the corresponding lanes was absence because the 5). Taking the percent of positive transfected cells and MFI
staining reagent was inaccessible to stain DNA.35,48)
into consideration, we finally chose lipid/DOPE ratio of 1:0.5
Optimization of Cationic Lipid/DOPE Ratios Neutral for T-C12-AH and lipid/DOPE ratio of 1:2 for T-C16-AH for
helper lipid DOPE plays an important role to form cationic further study.
liposomes. The appropriate addition of DOPE has been re-
Optimization of Cationic Lipids/DNA Ratios After the
ported to increase the gene delivery efficiency of cationic li- optimization of cationic lipid/DOPE ratio, we then determined
posomes.49–51) In an effort to research the influence of cationic the particle size and zeta potential of cationic liposomes/DNA
lipid/DOPE ratios on the capacity of gene delivery efficiency, lipoplexes and the transfection performance at different N/P
we prepared a series of cationic liposomes with different cat- ratios to investigate the effect of N/P ratio on transfection.
ionic lipid/DOPE molar ratios of 1:0.5, 1:1, 1:2, and 1:3 at As shown in Table 2, after binding with DNA at N/P ratio of
equal lipid/DNA molar ratio of 3:1. The transfection activity 1:1, the particle size of cationic liposomes/DNA lipoplexes
was evaluated through the percent of positive transfected cells increased when compared with free cationic liposomes, from
and MFI by flow cytometry in 293T cells as shown in Fig. 95.4 1.0nm to 132.9 1.6nm (T-C12-AH), and 79.6 1.1nm
4. The optimized lipid/DOPE ratio was found to be different to 195.4 0.9nm (T-C16-AH), respectively. With the increase
for the two cationic lipids. When the ratio of DOPE increased of cationic lipids (N/P ratios from 1:1 to 4:1), the particle
from 0.5 to 2, the gene delivery efficiency of lipid T-C16-AH size of lipoplexes became smaller and more compact because
increased. However, the gene delivery efficiency reduced with highly tight lipoplexes were formed with the increasing of N/P

Vol. 39, No. 7 (2016)
Biol. Pharm. Bull.
1117
Fig. 5. GFP Expression of T-C12-AH (A) and T-C16-AH (B) with Different Lipid/DOPE Molar Ratios in 293T Cells Observed by Fluorescence
Microscope
Table 2. Particle Sizes and Zeta Potentials of Cationic Liposome/DNA Lipoplexes at Optimal Cationic Lipid/DOPE Condition
Formulation
Liposome/DNA (molar ratio)
Size (nm)
Polydispersity (PDI)
Zeta potential (mV)
T-C12-AH
1:0
1:1
2:1
3:1
4:1
1:0
1:1
2:1
3:1
4:1
1:0
1:1
1:0
1:1
95.4 1.0
132.9 1.6
123.7 0.3
110.9 0.7
106.8 1.1
79.6 1.1
0.258 0.010
0.235 0.008
0.181 0.010
0.196 0.005
0.192 0.013
0.218 0.003
0.231 0.014
0.134 0.006
0.154 0.011
0.163 0.018
0.306 0.009
0.190 0.012
0.252 0.002
0.236 0.023
68.1 0.5
−17.8 0.2
25.4 0.9
28.0 1.6
34.6 0.5
61.9 4.4
−13.3 0.5
39.9 2.2
41.6 0.9
41.7 0.2
58.4 0.4
−38.5 0.6
60.4 0.7
−19.1 3.9
T-C16-AH
195.4 0.9
124.0 0.4
111.9 1.1
103.6 0.2
146.9 1.2
173.3 1.2
100.2 1.2
146.8 3.7
DC-Chol
DOTAP
ratio. We also measured the zeta potential of these lipoplexes 3 and mentioned above, when N/P ratio was over 1:1, DNA
and the control formulations. The lipoplexes showed negative was tightly compacted in the cationic liposomes, and the
zeta potentials for cationic lipids T-C12-AH or T-C16-AH at staining reagent was inaccessible to stain DNA thus leading
N/P ratio of 1, while zeta potentials turned to positive value to the absence of the fluorescene in the corresponding lanes.
at N/P ratio over 2. With the increase of cationic lipids, there The percent of positive transfected cells of T-C12-AH (47.7%)
were excess cationic lipids in the lipoplexes, thus resulting in was better than that of T-C16-AH (32.9%) at N/P ratio of 1:1,
the increasing of zeta potential. Under the optimal condition while the MFI showed no significant difference between these
of DC-Chol and DOTAP, the formulations also showed the two lipids at N/P ratio of 1:1 (Fig. 6). It was worth noting that
negative zeta potentials.
T-C12-AH exhibited higher gene transfection performance in
Lipoplexes at different N/P charge ratios were prepared and the percent of positive transfected cells than DC-Chol (15.8%)
examined for transfection activity in 293T cells. The results at N/P ratio of 1:1 and 2:1, and showed higher gene trans-
showed that both T-C12-AH and T-C16-AH at the N/P ratio fection activity in the MFI than DC-Chol at any N/P charge
of 1:1 showed highest gene transfection performance. Both ratios. T-C12-AH also exhibited higher gene transfection
cellular internalization and intracellular DNA release are performance in the percent of positive transfected cells than
common barriers for nonviral gene delivery system. Although DOTAP (24.7%) at N/P ratio of 1:1 and 2:1, while showed
positively charged lipoplexes are useful in the initial stages of comparable MFI with DOTAP at N/P ratio of 1:1, 2:1 and
endocytosis, complexes unpacking is generally assumed to be 3:1. For lipid T-C16-AH, it showed higher gene transfec-
necessary for DNA release and gene expression. The lipoplex- tion activity in the percent of positive transfected cells than
es with negative charge at the N/P ratio of 1:1 showed high DC-Chol only at N/P ratio of 1:1, while it exhibited higher
transfection efficiency because the lipoplexes at the higher MFI than DC-Chol at any N/P charge ratios. T-C16-AH also
N/P ratio compacted DNA too tightly to release DNA into cy- exhibited higher gene transfection performance in the percent
toplasm for further gene expression. The lipoplexes formed at of positive transfected cells than DOTAP at N/P ratio of 1:1,
high N/P ratio exhibited lower gene transfection performance and showed comparable MFI with DOTAP at N/P charge ra-
than that at N/P ratio of 1:1. This can also be confirmed by tios of 1:1 to 4:1. The images of GFP expression observed by
the results of agarose gel retardation assay, as shown in Fig. a fluorescence microscope were in line with the aboved results

1118
Biol. Pharm. Bull.
Vol. 39, No. 7 (2016)
Fig. 6. Transfection Activities of Cationic Lipids T-C12-AH (A) and T-C16-AH (B) at Various N/P Ratios in 293T Cells
The data of the percent of positive transfected cells (GFP Cell%) and mean fluorescence intensity (MFI) obtained from flow cytometry analysis are shown as the
mean S.D. (n=3). *p<0.05, **p<0.01, compared to DC-Chol. # p<0.05, ## p<0.01, compared to DOTAP. Ψ p<0.05, ΨΨ p<0.01, compared to T-C12-AH or T-C16-AH at
N/P ratio of 1:1. & p<0.01, compared to T-C12-AH at N/P ratio of 1:1.
Fig. 7. GFP Expression of Cationic Lipids T-C12-AH (A) and T-C16-AH (B) at Various N/P Ratios in 293T Cells Observed by Fluorescence Mi-
croscope
(Fig. 7). These results indicated that appropriate N/P ratio was
important for gene delivery efficiency.
The transfection activity of T-C12-AH and T-C16-AH
was also evaluated in HeLa cells at the optimized condi-
tion. As shown in Fig. 8, although the MFI of T-C12-AH
was lower than those of DC-Chol and DOTAP, T-C12-AH
showed higher gene transfection performance in the percent of
positive transfected cells than DOTAP and comparable gene
transfection performance with DC-Chol. T-C16-AH showed
lower gene transfection performance both in the percent of
positive transfected cells and MFI than that of DC-Chol, and
exhibited comparable gene transfection performance in the
percent of positive transfected cells with DOTAP. T-C12-
AH still showed higher gene transfection performance in the
percent of positive transfected cells than that of T-C16-AH in
HeLa cells. Considering the gene delivery efficiency both in
293T cells and HeLa cells, cationic lipid T-C12-AH showed
superior transfection activity.
Cytotoxicity Since good biocompatibility is of great
importance for gene carriers, the toxicity of cationic lipids
T-C12-AH and T-C16-AH was determined. The results
were shown as the percent of cell viability compared to the
control group. As shown in Fig. 9, both of these two lipids
showed lower toxicity than that of DC-Chol (IC50=49.9 µM)
Fig. 8. Transfection Activities of Cationic Lipids T-C12-AH and
T-C16-AH in HeLa Cells
The data of the percent of positive transfected cells (GFP Cell%) and mean fluo-
rescence intensity (MFI) obtained from flow cytometry analysis are shown as the
mean S.D. (n=3). *p<0.01, compared to DC-Chol. # p<0.01, compared to DOTAP.
& p<0.01, compared to T-C12-AH.

Vol. 39, No. 7 (2016)
Biol. Pharm. Bull.
1119
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