Synthesis and Characterization of Novel Cationic Lipids Derived from Thio Galactose
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- Qiao, W., Zhou, M. & Luo, L. J Surfact Deterg (2014) 17: 261. doi:10.1007/s11743-013-1474-0
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Two double chain cationic lipids QAS Cn-2-S (n = 12, 14) derived from thio galactose and carbamate-linkage tertiary amine were synthesized and their structures were confirmed by MS, TOF-MS, 1H NMR and 13C NMR. The QAS C12-2-S revealed superior surface activity compared with QAS C14-2-S with lower CMC and γCMC. Though Lipo C12-2-S displayed large average particle-size with high polydispersity, positive charged Lipo Cn-2-S can be combined with the negative charged DNA, also negatively stained TEM images confirmed the formation of vesicles. All the above prove that the Lipo Cn-2-S is helpful for gene transfection.
KeywordsSynthesisThio galactoseCationic lipidsVesicle
As one of the significant means of cancer treatment, gene therapy has received much attention for decades. It is crucial to find a carrier to enhance transfection efficiency with the in-depth research of this technology.
Cationic liposome–DNA complexes are attracting considerable attention as gene vectors due to their safety and other inherent advantages over viral delivery methods [1–3]. These advantages include ease and variability of preparation, lack of immunogenicity, and a capacity for DNA of unlimited size, allowing for delivery of artificial human chromosomes . However, cationic liposomes have low transfection efficiency in comparison with vital carriers. The transfection efficiency could be enhanced owing to the specific binding of ligands and receptors. As we all know, there is rich asialoglycoprotein on the surface of the liver organ, which as a receptor can specially recognize the compounds whose terminal part has non-reductive galactose and GalNAc . It has been confirmed that the galactose receptor-mediated pathway is the best for the hepatocytes in the all receptor-mediated pathways . The liver targeting and transfection efficiency would be significantly improved by the galactose-modified drug  or polymer carriers  in this way.
d-Galactose was purchased from the Sinopharm Chemical Reagent Co. Ltd., China. Thiourea and Potassium metabisulfite were purchased from Guangdong Shantou Xilong Chemical Factory, China. 1,2-Dibromoethane was obtained from Chengdu Kelong Chemical Reagent Factory, China. Potassium carbonate, sodium sulfate and sodium methoxide were purchased from Tianjin Fuchen Chemical Plant, China. D113 cation exchange resin was purchased from Shenyang Xinxing Reagent Factory, China. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Sigma-Aldrich (USA). Dichloromethane, chloroform, methanol, ethanol, hexane, ethyl acetate, and acetone were of analytical grade. Deionized water was used in all measurements.
The mass spectrum was obtained with Liquid chromatography/quadrupole time of flight tandem mass spectrometry (Micromass Britain, LC/Q-TOF-MS) and High Performance Liquid Chromatography/Mass selective Detector (HP, America, Model HP 1100LC/MSD). 1H-NMR and 13C-NMR spectra were recorded on a Bruker 400-MHz instrument (Bruker, Switzerland, Model AVANCE II 400 MHz). Melting points were tested with a micro-melting point apparatus (Ningbo Yongxin Optical Co., Ltd., China, Model X-4). The surface tension was determined with a processor tensiometer (Bowing Industry Corporation, America, Model TX-500C) by the spinning drop principle. Particle sizes and Zeta potentials were measured by nano particle size and zeta potential measurement (Zetasizer nano series Nano-ZS90, Malvern, UK). Vesicles were observed with a transmission electron microscope (JEOL, Japan, Model JEM-1200EX).
Preparation of 2,3,4,6-Tetra-O-acetyl-α-d-galactopyranosyl bromide (1)
2,3,4,6-Tetra-O-acetyl-α-d-galactopyranosyl bromide was prepared according to Ref. .
Preparation of 2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosylthiol (2)
Compound 1 (6.00 g, 14.63 mmol) was dissolved in anhydrous acetone (7 mL), thiourea (1.17 g, 14.63 mmol) was added and the mixture was refluxed for 1 h. After a rapid removal of acetone, the residue was dissolved in water (10 mL) and CH2Cl2 (10 mL). Potassium metabisulfite (2.59 g, 11.88 mmol) was now added and the mixture was refluxed for another 15 h. The reaction was monitored by thin layer chromatography (hexane: ethyl acetate = 5:2, v/v). The solution was then cooled down to r.t., and the mixture was extracted with CH2Cl2 twice. The dichloromethane extracts were combined, dried over anhydrous sodium sulfate, evaporated in vacuo and the resulting residue was subjected to column chromatography (hexane: ethyl acetate, 5:2, v/v) to obtain the white crystals m.p. 81.6–84.5 °C, in a 49.6 % yield.
1H NMR (400 MHz, MeOD, δ ppm): 1.99,2.07,2.08,2.17 (12H, 4 × s, 4 × CH3COO (Gal)), 2.36,2.39 (1H, d, J = 12.0, Gal-SH), 3.94–3.97 (1H, m, H-5(Gal)), 4.12–4.14 (2H, d, J = 6.8, H-6,6′(Gal)), 4.52–4.56 (1H, t, H-1(Gal)), 5.01–5.04 (1H, dd, J1 = 10.0, J2 = 3.2, H-2(Gal)), 5.16–5.21 (1H, t, H-3(Gal)), 5.43–5.44 (1H, dd, J1 = 3.2, J2 = 0.8, H-4(Gal)), 7.28 (s, CDCl3);
13C NMR (400 MHz, MeOD, δ ppm):169.84,169.97,170.16,170.38 (C=O(Gal)), 79.19 (C-1(Gal)), 76.73–77.37(CDCl3), 67.24,70.84,71.57,74.95 (C-2,3,4,5(Gal)), 61.47 (C-6(Gal)), 20.56,20.67,20.69,20.83 (CH3COO(Gal));
MS (positive): m/z 387.1420 [M + Na]+.
Preparation of 2-Bromoethyl-2,3,4,6-tetra-O-acetyl-β-d-thiogalactopyranoside (3)
A mixture of compound 2 (2.00 g, 5.49 mmol) and potassium carbonate (800 mg, 5.80 mmol) in acetone–water (4:3, 14 mL) was stirred under nitrogen. Then, 1,2-dibromoethane (3.6 mL, 7.85 mmol) was added and the reaction was stirred at room temperature. The reaction was monitored by thin layer chromatography with hexane: ethyl acetate (2:1, v/v) as eluent. The mixture was extracted twice with CH2Cl2 (50 mL). The combined organic layer was dried with anhydrous sodium sulfate and concentrated to give a yellow oil. The residue was purified by silica gel chromatography using hexane and ethyl acetate (2:1, v/v) as the eluent to give the bromide intermediate as white crystals m.p. 71.8–74.5 °C, in a 44.6 % yield.
1H NMR (400 MHz, MeOD, δ ppm): 1.99,2.05,2.09,2.17 (12H, 4 × s, 4 × CH3COO(Gal)), 2.97–3.23 (2H, m, S-CH2, in which 2.97–3.05 (1H, m), 3.17–3.25 (1H, m)), 3.52–3.61 (2H, m, CH2Br), 3.94–3.97(1H, t, H-5(Gal)), 4.08–4.17 (2H, m, H-6,6′(Gal)), 4.54–4.56 (1H, d, J = 8.0, H-1(Gal)), 5.03–5.07 (1H, dd, J1 = 12.0, J2 = 4.0, H-2(Gal)), 5.22–5.27 (1H, t, H-3(Gal)), 5.44–5.45 (1H, d, J = 2.4, H-4(Gal)), 7.28 (s, CDCl3);
13C NMR (400 MHz, MeOD, δ ppm): 169.56,169.98,170.13,170.41 (C=O(Gal)), 84.51 (C-1(Gal)), 76.73–77.36(CDCl3), 67.00,67.24,71.70,74.72 (C-2,3,4,5(Gal)), 61.69 (C-6(Gal)), 32.81(CH2Br), 30.64(S-CH2), 20.57,20.67,20.70,20.77 (CH3COO(Gal));
MS (positive): m/z [M + Na]+ 495.0378 (1Br) [2 M + Na]+ 967.0661.
Preparation of 3-(Dimethylamino)-propane-1,2-di-alkylcarbamate (4)
Tertiary amines with carbamate as the important intermediates were synthesized according to the literature procedures .
Preparation of 2,3-Bis(dodecylcarbamoyloxy)-N,N-dimethyl-N-[2-(2,3,4,6-tetra-O-acetyl-1-thio-β-d-galactopyranosyl) Ethyl] Propanyl Ammonium Bromide (5)
Anhydrous compound 3 (1.25 g, 2.66 mmol) and 3-(dimethylamino) propanyl-1,2-didodecylcarbamate (1.5 g, 2.67 mmol) were dissolved in anhydrous ethanol (15 mL). The mixture was refluxed for 48 h under a stream of nitrogen. The solvent was evaporated in vacuo and the resulting residue was subjected to silica gel column chromatography ([CHCl3/NH3 = 5/1]/CH3OH = 10/1, v/v). Compound 5 was obtained as a light yellow viscous fluid with a 30 % yield.
1H NMR (400 MHz, MeOD, δ ppm): 0.88,0.90,0.92 (6H, t, 2 × CH2CH3), 1.29 (36H, s, 2 × (CH2)9), 1.49,1.51 (4H, 2 × s, 2 × NHCH2CH2), 1.96,2.05,2.06,2.16 (12H, 4 × s, 4 × CH3COO(Gal)), 3.08–3.26 (12H, m, 2 × NHCH2, SCH2(Et-Linker), 2 × N+CH3), 3.31 (s, MeOD), 3.69–3.79 (4H, m, 2 × N+CH2), 4.10–4.42 (5H, m, H-5,6,6′(Gal), NHCOOCH2), 4.88 (s, H2O), 4.92–4.94 (1H, d, J = 8.0, H-1(Gal)), 5.15–5.23 (2H, m, H-2,3(Gal)), 5.48–5.52 (2H, m, H-4(Gal), NHCOOCH);
13C NMR (400 MHz, MeOD, δ ppm): 171.28,171.53,171.80,172.22 (C=O(Gal)), 156.84,157.87 (NHC=O), 83.96,84.86 (C-1, α/β(Gal)), 68.37,69.17,72.95,76.43 (C-2,3,4,5(Gal)), 67.58 (NHCOOCH), 65.21,66.45 (N+(CH2)2), 64.93 (NHCOOCH2), 62.86 (C-6(Gal)), 52.61,52.87 (N+(CH3)2), 49.00 (MeOD), 41.99,42.11 (NHCH2), 22.95–33.08 ((CH2)10, SCH2(Et-Linker)), 20.48,20.57,20.69,20.80 (CH3COO(Gal)), 14.44 (CH3CH2);
MS (positive): m/z 932.4318 [M−Br]+.
Preparation of 2,3-Bis(dodecylcarbamoyloxy)-N,N-dimethyl-N-[2-(1-thio-β-d-galactopyranosyl) Ethyl] Propanyl Ammonium Bromide (6, denoted as QAS C12-2-S)
Compound 5 (0.83 g, 0.82 mmol) was dissolved in anhydrous ethanol (15 mL). Sodium methoxide (0.31 g, 5.74 mmol) was added and the mixture was stirred for 1 h. D113 cation exchange resins were added when TLC (CHCl3/CH3OH = 4/1, v/v) showed the above reaction was completed. Until the pH of the solution had changed to neutral, chloroform (5 mL) was added to distill the mixture. The solvent was removed by rotary evaporation after filtration. Compound 6 was obtained as a light yellow solid with a 100 % yield.
1H NMR (400 MHz, MeOD, δ ppm): 0.88,0.90,0.92 (6H, t, 2 × CH2CH3), 1.29 (36H, s, 2 × (CH2)9), 1.49,1.51 (4H, 2 × s, 2 × NHCH2CH2), 3.02–3.26 (12H, m, 2 × NHCH2 2 × N+CH3, OCH2 (Et-Linker)), 3.31 (s, MeOD), 3.46–3.87 (10H, m, 2 × N+CH2, H-2,3,4,5,6,6′(Gal)), 4.06–4.11 (2H, m, NHCOOCH2) 4.42–4.46 (1H, dd, J1 = 12.0, J2 = 4.0, H-1(Gal)), 4.89 (s, H2O), 5.47–5.57 (1H, m, NHCOOCH), 8.55 (6H, s, 2 × NH, 6 × OH);
13C NMR (400 MHz, MeOD, δ ppm): 155.44,156.58 (NHC=O), 85.66,86.36 (C-1, α/β(Gal)), 69.38,69.84,74.66,79.60 (C-2,3,4,5(Gal)), 66.30 (NHCOOCH), 65.51,65.75 (N+CH2), 63.72 (NHCOOCH2), 61.83 (C-6(Gal)), 50.89,51.13 (N+(CH3)2), 49.00 (MeOD), 40.55,40.67 (NHCH2), 22.37–31.70 ((CH2)10, SCH2(Et-Linker)), 13.10 (CH3CH2);
MS (positive): m/z 764.7 [M−Br]+.
Preparation of 2,3-Bis(tetradecylcarbamoyloxy)-N,N-dimethyl-N-[2-(2,3,4,6-tetra-O-acetyl-1-thio-β-d-galactopyranosyl) Ethyl] Propanyl Ammonium Bromide (7)
Compound 7 was synthesized as the same procedure as compound 5 with a 33.3 % yield.
1H NMR (400 MHz, MeOD, δ ppm): 0.88,0.90,0.92 (6H, t, 2 × CH2CH3), 1.29 (44H, s, 2 × (CH2)11), 1.49,1.51 (4H, 2 × s, 2 × NHCH2CH2), 1.96,2.05,2.06,2.16 (12H, 4 × s, 4 × CH3COO(Gal)), 3.08–3.26 (12H, m, 2 × NHCH2, SCH2(Et-Linker), 2 × N+CH3), 3.30 (s, MeOD), 3.67–3.81 (4H, m, 2 × N+CH2), 4.06–4.41 (5H, m, H-5,6,6′(Gal), NHCOOCH2), 4.88 (s, H2O), 4.91–4.94 (1H, d, J = 8.8, H-1(Gal)), 5.15–5.23 (2H, m, H-2,3(Gal)), 5.48 (1H, s, H-4(Gal)), 5.51–5.52 (1H, d, J = 4.8, NHCOOCH), 7.90 (s, CHCl3);
13C NMR (400 MHz, MeOD, δ ppm): 171.27,171.44,171.52,171.79 (C=O(Gal)), 157.95,156.84 (NHC=O), 83.94,84.86 (C-1, α/β(Gal)), 79.47 (CHCl3) 68.35,69.15,72.94,76.42 (C-2,3,4,5(Gal)), 67.56 (NHCOOCH), 65.21,66.43 (N+(CH2)2), 64.87 (NHCOOCH2), 62.86 (C-6(Gal)), 52.59,52.87 (N+(CH3)2), 49.00 (MeOD), 41.98,42.10 (NHCH2), 22.94-33.08 ((CH2)12, SCH2(Et-Linker)), 20.57,20.70,20.72,20.81 (CH3COO(Gal)), 14.45 (CH3CH2);
MS (positive): m/z 988.7 [M−Br]+.
Preparation of 2,3-Bis(tetradecylcarbamoyloxy)-N,N-dimethyl-N-[2-(1-thio-β-d-galactopyranosyl) Ethyl] Propanyl Ammonium Bromide (8, denoted as QAS C14-2-S)
Compound 8 was synthesized as the same procedure as compound 6 with a 100 % yield.
1H NMR (400 MHz, MeOD, δ ppm): 0.88,0.89,0.91 (6H, t, 2 × CH2CH3), 1.28 (44H, s, 2 × (CH2)11), 1.49,1.50 (4H, 2 × s, 2 × NHCH2CH2), 1.96,2.15 (2 × s, OH), 3.04–3.17 (6H, m, 2 × NHCH2, SCH2(Et-Linker)), 3.20(6H, s, N+(CH3)2) 3.30 (s, MeOD), 3.47–3.88 (10H, m, 2 × N+CH2, H-2,3,4,5,6,6′(Gal)), 4.07–4.11,4.25–4.27 (2H, m, NHCOOCH2) 4.45–4.48 (1H, dd, J1 = 9.6, J2 = 5.6, H-1(Gal)), 4.84 (s, H2O), 5.48–5.56 (1H, m, NHCOOCH), 7.89 (s, CHCl3), 8.48 (s, NH);
13C NMR (400 MHz, MeOD, δ ppm): 156.84,157.96 (NHC=O), 87.01,87.69 (C-1, α/β(Gal)), 81.03(CHCl3), 67.71,70.76,71.28,76.09 (C-2,3,4,5(Gal)), 67.22 (NHCOOCH), 65.16,66.98 (N+CH2), 63.25 (NHCOOCH2), 63.18 (C-6(Gal)), 52.29,52.53 (N+(CH3)2), 49.00 (MeOD), 41.95,42.08 (NHCH2), 23.29–33.07 ((CH2)12, SCH2 (Et-Linker)), 14.45 (CH3CH2);
MS (positive): m/z 820.4453 [M−Br]+.
Equilibrium Surface Tension
where, C1, C2 and C3 are the amount of carbon, methylene and methyl, respectively.
Size and Zeta Potential Measurements
In the formulation of cationic liposomes containing DOPE as a helper lipid, cationic lipid/DOPE at a molar ratio of 1:1 was prepared by conventional thin film evaporation and an ultrasonic method [12, 13]. The aqueous solutions of the cationic liposomes were sonicated in the ultrasonic bath. The liposomes were obtained by filtration through the 200 nm membrane, final concentration of which was 1 mg/mL. Particle sizes and zeta potentials were measured at r.t. by Nanoparticle Size and Zeta Potential Measurement.
Transmission Electron Microscopy
Observation by transmission electron microscope (TEM) was performed with a negative staining method. A drop of solution was placed on a Formvar covered TEM grid (copper grid, 3.02 mm, 200 mesh) and stained with a drop of 2 wt% phosphotungstic acid aqueous solution. The excess solution was removed by blotting with a filter paper. TEM, operating at 80.5 kV, was used to investigate the samples.
Results and Discussion
CMC, γCMC, Γm, Am and P
Physicochemical properties of the surfactants QAS Cn-2-S(O)
2.4 × 10−4
7.07 × 10−10
4.5 × 10−6
8.28 × 10−10
4.0 × 10−5
5.21 × 10−10
Critical packing parameter of the surfactants QAS Cn-2-S
It is well known that the CMCs of conventional ionic surfactants decrease with the increase of the carbon number of the hydrophobic chain up to 16 . In this research, CMCs of QAS Cn-2-S increased from 4.5 × 10−6 to 4.0 × 10−5 mol·L−1 with increasing the hydrophobic chain from 12 to 14, which is not consistent with the tendency of the common homologous. Generally, hydrocarbon chain of the surfactant demonstrates weak affinity with water molecules due to its hydrophobicity, thus making the interfacial free energy between the hydrophobic hydrocarbon chain and water much higher. In order to reduce this high interfacial free energy, the hydrophobic hydrocarbon chain often remains in a coiled state. When the length of the hydrophobic hydrocarbon chain exceeds a certain value, the coiled and disorder state of the hydrophobic chain will be further strengthened, so that it hinders the self-assembly process of the surfactant molecules and the formation of micelles, thus giving higher CMC .
It is of vital importance whether vesicles could be formed or not for QAS Cn-2-S as a gene delivery vehicle in future. Tanford  and Israelachvili  raised that the critical packing parameter P predicts the formation of various surfactant. Different P values are compatible with different geometric shapes of the aggregates. When P is less than 1/3, spherical micelles are the preferred form of aggregation, as the ratio of surfactant head group area is large in comparison to the hydrophobic part. Cylindrical micelles form when P is between 1/3 and 1/2. When P is greater than 1/2, first formed are highly curved bilayer vesicles and then flat bilayers as P goes to 1. The structure of QAS Cn-2-S is single head and two hydrophobic carbon chains. As for QAS C12-2-S, P is 0.98, which indicates flat bilayers are formed. In addition, the P value of QAS C14-2-S is 0.62, which shows curved bilayer vesicles are energetically favored.
Particle Size and Zeta-Potential
Particle-size refers to the equivalent diameter of the particles of the cationic liposomes. Zeta-potential is the potential of the shear plane, which is an important indicator to characterize the stability of colloidal dispersions. Positively charged cationic liposomes can be combined with the negatively charged DNA to achieve the purpose of gene transfection. As a result, the particle size and zeta potential of cationic liposomes directly affect their transfection efficiency.
Size and Zeta-potential of liposomes
Formation of Vesicles
The left presented the different sizes of C12-2-S/DOPE liposome, mainly large particle-size vesicles. In addition, the right illustrated the vesicle formation of C14-2-S/DOPE liposome. Evidently, Lipo C14-2-S displayed ones of smaller size and better polydispersity compared to Lipo C12-2-S, which was consistent with the results of size measurement. According to the critical packing parameter P, QAS C14-2-S was inclined to form vesicles and QAS C12-2-S favored flat bilayers, which lead to the size of Lipo C14-2-S much smaller. In the aqueous system, the average particle size of liposomes showed comparatively large due to vesicles aggregation.
Two double chain cationic lipids Cn-2-S derived from thio galactose were synthesized and confirmed by MS, TOF-MS, 1H NMR and 13C NMR. QAS C12-2-S appears lower CMC value and γCMC than QAS C14-2-S counterpart. Vesicles are formed in aqueous solution and it is consistent with the result of the critical packing parameter. Moreover, negatively stained TEM images confirmed the formation of vesicles.
The authors are grateful for the financial support from the National Natural Science Foundation of China (20876023).