Materials and instruments
All reagents were purchased from commercial sources and were used as received. Acetonitrile, dichloromethane, acetone, and N,N-dimethyl formamide (DMF) were dried and purified under nitrogen by using standard methods and were distilled immediately before use.
The HepG2 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Solarbio Company. The enhanced green fluorescent protein (eGFP) encoding plasmid DNA (pDNA) was purchased from Aldevron and cloned in Escherichia coli-DH5a (TIANGEN)and then was extracted with QIAGEN Mega Plasmid kit (Qiagen EndoFree Plasmid, Sigma-Aldrich) according to the manufacturer’s protocol. Label IT Cy5 Labeling Kit was purchased from Mirus Bio Company. Hoechst 33342 was purchased from Molecular Probes Company. Ultrapure milli-Q water was used in all DNA condensation assays.
All chemical reactions were monitored by TLC on silica gel GF 254. Spots were detected under UV light and/or by charring with potassium permanganate. Column chromatography was performed on silica gel (100–200 μm).1H NMR and 13C NMR spectra were recorded on Bruker ascended 600M AVANCEⅢ HD. Mass spectra (ESI) were acquired on Thermo Finnigan. Electrophoresis was operated using a submarine system of BIO-RAD Power Pac TM Universal Power Supply. Plasmid concentration was measured by Nanodrop (Thermo Scientific 2000C). Hydrodynamic diameters and zeta potential were determined using a Nano-ZS 90 Laser Particle Size and Zeta Potential Analyzer. The cell fluorescences were observed using a Leica biological microscope DMI 4000B.
1. Sulfonium compound synthesis and structure characterization
General procedure for the preparation of compounds P
To a solution of triethylene glycol or tetraethylene glycol (1 eq) and alkyl iodide (1 eq) in DMF (40 mL) at 0 ºC was added NaH (1.5 eq, 60% in mineral oil). After stirring for 2 h, the reagents were quenched by the addition of H2O. The solution was extracted with Et2O twice and the organic layer was washed with brine, dried (MgSO4), and concentrated. The residue was purified by chromatography (ethyl acetate–hexane) to give P colorless oil. The product was used in the next step without characterization.
General procedure for the preparation of compound Q
The mono alcohol P (1.0 equiv.) was dissolved in CH2Cl2 (10 mL) at 0 ºC before Et3N (1 mL) and ethanesulfonyl chloride (1.2 equiv.) were added. The mixture was stirred for 30 min at r.t. and then treated with H2O (10 mL). The solution was extracted with CH2Cl2 (10 mLÎ2). The organic layer was washed with brine, dried (MgSO4), and concentrated to give the crude sulfonate which was dissolved in acetone (10 mL) and then fused NaI (1.5 equiv.) was added. The reaction mixture was heated at 50 ºC for 4 h, before being cooled and concentrated. The resulting residue was purified by chromatography (ethyl acetate–hexane) to give the corresponding iodide Q. The compound was used in the next step without characterization.
General procedure for the preparation of A (sulfonium lipid).
To a solution of alkyl iodide Q (1.0 equiv.) in dry CH3CN (10 mL) was added Tetrahydrothiophene or Tetrahydrothiopyrane (1.5 equiv.) and AgBF4 (1.5 equiv.). The reaction mixture was covered with aluminum foil and refluxed at 85 ºC for 24 h. The mixture was cooled to rt and filtered. The filtrate was treated with ion exchange resin (Cl-) for 2 h and then filtered, concentrated and the resulting residue was purified by chromatography (toluene–methanol) to give the sulfonium lipid A.
2-(2-(2-(nonyloxy) ethoxy) ethoxy)ethanol (P1)
In the general procedure for preparing P, triethylene glycol (2 mL, 14.93 mmol) was treated with 1-iodononane (3.79 g, 14.93 mmol) and NaH (0.90 g, 22.5 mmol)to form P1 (1.93 g, 7.01 mmol, yield 47%) as colorless oil, Rf = 0.43 (ethyl acetate-hexane 3:2).
2-(2-(2-(dodecyloxy) ethoxy) ethoxy)ethanol (P2)
In the general procedure for preparing P, triethylene glycol (2 mL, 14.93 mmol) was treated with 1-iodododecane (4.42 g, 14.93 mmol) and NaH (0.90 g, 22.5 mmol)to form P2 (1.89 g, 5.95 mmol, yield 40%) as colorless oil, Rf = 0.41 (ethyl acetate-hexane 3:2).
2-(2-(2-(hexadecyloxy) ethoxy)ethoxy)ethanol (P3)
In the general procedure for preparing P, triethylene glycol (2 mL, 14.93 mmol) was treated with 1-iodohexadecane (5.26 g, 14.93 mmol) and NaH (0.90 g, 22.5 mmol)to form P3 (1.98 g, 5.52 mmol, yield 37%) as colorless oil, Rf = 0.38 (ethyl acetate-hexane 3:2).
2-(2-(2-(2-(nonyloxy)ethoxy)ethoxy) ethoxy)ethanol (P4)
In the general procedure for preparing P, tetraethylene glycol (2 mL, 11.59 mmol) was treated with 1-iodononane (3.79 g, 14.93 mmol) and NaH (0.70 g, 17.38 mmol)to form P4 (1.78 g, 5.56 mmol, yield 48%) as colorless oil, Rf = 0.43 (ethyl acetate-hexane 3:2).
2-(2-(2-(2-( dodecyloxy)ethoxy)ethoxy) ethoxy)ethanol (P5)
In the general procedure for preparing P, tetraethylene glycol (2 mL, 11.59 mmol) was treated with 1-iodododecane (4.42 g, 11.59 mmol) and NaH (0.70 g, 17.38 mmol)to form P5 (2.18 g, 6.02 mmol, yield 52%) as colorless oil, Rf = 0.41 (ethyl acetate-hexane 3:2).
2-(2-(2-(2-(hexadecyloxy)ethoxy)ethoxy) ethoxy)ethanol (P6)
In the general procedure for preparing P, tetraethylene glycol (2 mL, 11.59 mmol) was treated with 1-iodohexadecane (5.26 g, 11.59 mmol) and NaH (0.70 g, 17.38 mmol)to form P6 (1.70 g, 4.06 mmol, yield 35%) as colorless oil, Rf = 0.38 (ethyl acetate-hexane 3:2).
1-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)nonane (Q1)
In the general procedure for preparing Q, P1 (1.93 g, 7.01 mmol) was treated with ethanesulfonyl chloride (1.00 mL, 10.52 mmol), to form a sulfonate intermediate which was reacted with NaI (1.58 g, 10.52 mmol) to form Q1 (1.99 g, 5.19 mmol) as yellowish oil, yield 74%, Rf = 0.35 (ethyl acetate-hexane 1:4).
1-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)dodecane (Q2)
In the general procedure for preparing Q, P2 (1.89 g, 5.95 mmol) was treated with ethanesulfonyl chloride (0.93 mL, 8.93 mmol), to form a sulfonate intermediate which was reacted with NaI (1.34 g, 8.93 mmol) to form Q2 (1.50 g, 3.51 mmol) as yellowish oil, yield 59%, Rf = 0.37 (ethyl acetate-hexane 1:4).
1-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)hexadecane (Q3)
In the general procedure for preparing Q, P3 (1.98 g, 5.52 mmol) was treated with ethanesulfonyl chloride (0.79 mL, 8.28 mmol), to form a sulfonate intermediate which was reacted with NaI (1.24 g, 8.28 mmol) to form Q3 (1.15 g, 2.37 mmol) as yellowish oil, yield 43%, Rf = 0.33 (ethyl acetate-hexane 1:4).
1-(2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethoxy)nonane (Q4)
In the general procedure for preparing Q, P4 (1.78 g, 5.56 mmol) was treated with ethanesulfonyl chloride (0.79 mL, 8.34 mmol), to form a sulfonate intermediate which was reacted with NaI (1.25 g, 8.28 mmol) to form Q4 (1.36 g, 3.17mmol) as yellowish oil, yield 57%, Rf = 0.36 (ethyl acetate-hexane 1:4).
1-(2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethoxy)dodecane (Q5)
In the general procedure for preparing Q, P5 (2.18 g, 6.02 mmol) was treated with ethanesulfonyl chloride (0.86 mL, 9.03 mmol), to form a sulfonate intermediate which was reacted with NaI (1.35 g, 9.03 mmol) to form Q5 (1.56 g, 3.31 mmol) as yellowish oil, yield 55%, Rf = 0.38 (ethyl acetate-hexane 1:4).
1-(2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethoxy) hexadecane (Q6)
In the general procedure for preparing Q, P6 (1.70 g, 4.06 mmol) was treated with ethanesulfonyl chloride (0.58 mL, 6.09 mmol), to form a sulfonate intermediate which was reacted with NaI (0.91 g, 6.09 mmol) to form Q6 (1.24 g, 2.35 mmol) as yellowish oil, yield 58%, Rf = 0.40 (ethyl acetate-hexane 1:4).
S-(2-(2-(2-(nonyloxy)ethoxy)ethoxy)ethyl-tetrahydro-thiophenium chloride (A1)
In the general procedure for preparing A, Q1 (0.93 g, 2.43 mmol) was reacted with tetrahydrothiophene (0.32 mL, 3.65 mmol) and AgBF4 (0.71 g, 3.65 mmol) to form A1 (0.58 g, 1.68 mmol) as yellowish oil, yield 69%, Rf = 0.32 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.90 (t, J = 4.8 Hz, 2 H, CH2), 3.64-3.63 (m, 2 H, CH2), 3.58-3.56 (m, 6 H, 3 CH2), 3.51-3.48 (m, 4 H, 2 CH2), 3.44-3.33 (m, 4 H, 2 CH2), 2.31-2.30 (m, 2 H, SCH2CH2), 2.24-2.22 (m, 2 H, SCH2CH2), 1.52 (q, J = 6.3 Hz, 2 H, CH2), 1.29 (m, 12 H, 6 CH2), 0.88 (t, J = 6.3 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.33, 70.90, 70.67, 70.30, 70.26, 65.87, 44.43 (2 C), 43.35, 32.14, 29.98, 29.84, 29.73, 29.55, 28.82 (2 C), 26.41, 22.91, 13.95, HR-MS (ESI) m/z: Calcd for C19H39O3S {[M-Cl-]+} 347.2614, found: 347.2896.
S-(2-(2-(2-(nonyloxy)ethoxy)ethoxy)ethyl-tetrahydro-thiopyranium chloride (A2)
In the general procedure for preparing A, Q1 (1.06 g, 2.67 mmol) was reacted with tetrahydrothiopyrane (0.43 mL, 4.01 mmol) and AgBF4 (0.78 g, 4.01 mmol) to form A2 (0.61 g, 1.68 mmol) as yellowish oil, yield 63%, Rf = 0.37 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.99 (t, J = 5.4 Hz, 2 H, CH2), 3.64-3.62 (m, 2 H, CH2), 3.57-3.54 (m, 2 H, CH2), 3.55-3.54 (m, 4 H, 2 CH2), 3.50-3.49 (m, 2 H, CH2), 3.40 (t, J = 6.6 Hz, 2 H, CH2), 3.30 (m, 2 H, CH2), 2.17-2.11 (m, 2 H, CH2), 1.96-1.94 (m, 2 H, CH2), 1.90-1.83 (m, 2 H, CH2), 1.76-1.70 (m, 1 H, CH2), 1.67-1.60 (m, 1 H, CH2), 1.52 (q, J = 6.9 Hz, 2 H, CH2), 1.28 (m, 12 H, 6 CH2), 0.88 (t, J = 6.9 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.33, 70.81, 70.66, 70.29, 70.27, 65.49, 37.24(2 C), 32.15, 30.00, 29.85, 29.75, 29.57, 26.44, 22.92(2 C), 22.83, 21.50(2 C), 13.97, HR-MS (ESI) m/z: Calcd for C20H41O3S {[M-Cl-]+} 361.2771, found: 361.3073.
S-(2-(2-(2-(dodecyloxy)ethoxy)ethoxy)ethyl-tetrahydro-thiophenium chloride (A3)
In the general procedure for preparing A, Q2 (0.79 g, 1.86 mmol) was reacted with tetrahydrothiophene (0.25 mL, 2.79 mmol) and AgBF4 (0.54 g, 2.79 mmol) to form A3 (0.42 g, 1.08 mmol) as yellowish oil, yield 58%, Rf = 0.35 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.93 (t, J = 5.4 Hz, 2 H, CH2), 3.65-3.62 (m, 4 H, 2 CH2), 3.59-3.55 (m, 6 H, 3 CH2), 3.53-3.52 (m, 4 H, 2CH2), 3.40 (t, J = 6.6 Hz, 2 H, CH2), 2.35-2.32 (m, 2 H, SCH2CH2), 2.28-2.23 (m, 2 H, SCH2CH2), 1.52 (q, J = 6.3 Hz, 2 H, CH2), 1.28 (m, 18 H, 9 CH2), 0.88 (t, J = 6.3 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.26, 70.87, 70.64, 70.28, 70.25, 65.92, 44.59 (2 C), 43.38, 32.10, 29.95, 29.84, 29.82, 29.81, 29.67, 29.58, 29.53, 28.90, 26.36, 26.34, 22.85, 13.89, HR-MS (ESI) m/z: Calcd for C22H45O3S {[M-Cl-]+} 389.3084, found: 390.3174.
S-(2-(2-(2-(dodecyloxy)ethoxy)ethoxy)ethyl-tetrahydro-thiopyranium chloride (A4),
In the general procedure for preparing A, Q2 (0.71 g, 1.65 mmol) was reacted with tetrahydrothiopyrane (0.26 mL, 2.48 mmol) and AgBF4 (0.48 g, 2.48 mmol) to form A4 (0.41 g, 1.01mmol) as yellowish oil, yield 69%, Rf = 0.36 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.96 (t, J = 5.4 Hz, 2 H, CH2), 3.66-3.65 (m, 2 H, CH2), 3.63-3.60 (m, 4 H, 2 CH2), 3.58-3.56 (m, 4 H, 2 CH2), 3.53-3.52 (m, 2 H, CH2), 3.42 (t, J = 6.9 Hz, 2 H, CH2), 3.37 (m, 2 H, CH2), 2.18-2.14 (m, 2 H, CH2), 1.93-1.86 (m, 2 H, CH2), 1.79-1.73 (m, 1 H, CH2), 1.71-1.64 (m, 1 H, CH2), 1.54 (q, J = 6.9 Hz, 2 H, CH2), 1.30 (m, 18 H, 9 CH2), 0.91 (t, J = 6.9 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.34, 70.86, 70.69, 70.31, 70.29, 65.51, 37.27 (2 C), 32.17, 30.02, 29.92, 29.89 (4 C), 29.74, 29.61, 26.44, 22.93, 22.84, 21.49 (2 C), 13.93, HR-MS (ESI) m/z: Calcd for C23H47O3S {[M-Cl-]+} 403.3240, found: 403.3463.
S-(2-(2-(2-(hexadecyloxy)ethoxy)ethoxy)ethyl-tetrahydro-thiophenium chloride (A5)
In the general procedure for preparing A, Q3 (0.56 g, 1.15 mmol) was reacted with tetrahydrothiophene (0.15 mL, 1.73 mmol) and AgBF4 (0.34 g, 1.73 mmol) to form A5 (0.24 g, 0.54 mmol) as yellowish oil, yield 47%, Rf = 0.35 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.89 (t, J = 5.4 Hz, 2 H, CH2), 3.64-3.62 (m, 2 H, CH2), 3.58-3.57 (m, 2 H, CH2), 3.56-3.54 (m, 2 H, CH2), 3.52-3.50 (m, 4 H, 2 CH2), 3.48-3.43 (m, 2 H, CH2), 3.40 (t, J = 6.9 Hz, 2 H, CH2), 3.36 (t, J = 5.4 Hz, 2 H, CH2), 2.33-2.28 (m, 2 H, SCH2CH2), 2.23-2.19 (m, 2 H, SCH2CH2), 1.52 (q, J = 7.2 Hz, 2 H, CH2), 1.27 (m, 26 H, 13 CH2), 0.88 (t, J = 7.2 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.38, 70.97, 70.74, 70.38, 70.34, 65.90, 44.41 (2 C), 43.42, 32.22, 30.06, 29.96(5 C), 29.94 (3 C), 29.79, 29.66, 28.83 (2 C), 26.48, 22.97, 13.99, HR-MS (ESI) m/z: Calcd for C26H53O3S {[M-Cl-]+} 445.3710, found: 445.4105.
S-(2-(2-(2-(hexadecyloxy)ethoxy)ethoxy)ethyl-tetrahydro-thiopyranium chloride (A6)
In the general procedure for preparing A, Q3 (0.59 g, 1.22 mmol) was reacted with tetrahydrothiopyrane (0.20 mL, 1.83 mmol) and AgBF4 (0.36 g, 1.83 mmol) to form A6 (0.29 g, 0.63 mmol) as yellowish oil, yield 62%, Rf = 0.32 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.93 (t, J = 5.4 Hz, 2 H, CH2), 3.66-3.64 (m, 2 H, CH2), 3.60-3.59 (m, 2 H, CH2), 3.58-3.56 (m, 2 H, CH2), 3.53-3.48 (m, 6 H, 3 CH2), 3.42 (t, J = 6.9 Hz, 2 H, CH2), 3.23 (m, 2 H, CH2), 2.17-2.13 (m, 2 H, CH2), 1.92-1.85 (m, 2 H, CH2), 1.78-1.72 (m, 1 H, CH2), 1.67-1.61 (m, 1 H, CH2), 1.54 (q, J = 6 Hz, 2 H, CH2), 1.30 (m, 26 H, 13 CH2), 0.95-0.90 (m, 3 H, CH3), 13C NMR (CD3CN, δ): 71.38, 70.89, 70.72, 70.34, 70.33, 65.48, 37.37 (2 C), 32.22, 30.06, 29.96 (5 C), 29.93 (4 C), 29.79, 29.66, 26.49, 22.97, 22.91, 21.45 (2 C), 13.98, HR-MS (ESI) m/z: Calcd for C27H55O3S {[M-Cl-]+} 459.3866, found: 459.4135.
S-(2-(2-(2-(2-(nonyloxy)ethoxy)ethoxy)ethoxy)ethyl-tetrahydro-thiophenium chloride(A7)
In the general procedure for preparing A, Q4 (0.68 g, 1.15 mmol) was reacted with tetrahydrothiophene (0.21 mL, 2.37 mmol) and AgBF4 (0.46 g, 2.37 mmol) to form A7 (0.43 g, 1.09 mmol) as yellowish oil, yield 69%, Rf = 0.35 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.90 (t, J = 5.4 Hz, 2 H, CH2), 3.64 (t, J = 4.2 Hz, 2 H, CH2), 3.60-3.54 (m, 10 H, 5 CH2), 3.51-3.49 (m, 4 H, 2 CH2), 3.41 (m, 4 H, 2 CH2), 2.32-2.27 (m, 2 H, SCH2CH2), 2.24-2.21 (m, 2 H, SCH2CH2), 1.52 (q, J = 6 Hz, 2 H, CH2), 1.29-1.28 (m, 12 H, 6 CH2), 0.88 (t, J = 6.6 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.36, 70.87, 70.64, 70.61, 79.59, 70.31, 70.22, 65.89, 44.42 (2 C), 43.36, 32.17, 29.96, 29.87, 29.76, 29.58, 28.83 (2 C), 26.43, 22.94, 13.98, HR-MS (ESI) m/z: Calcd for C21H43O4S {[M-Cl-]+} 391.2877, found: 391.2936.
S-(2-(2-(2-(2-(nonyloxy)ethoxy)ethoxy)ethoxy)ethyl-tetrahydro-thiopyranium chloride (A8)
In the general procedure for preparing A, Q4 (0.68 g, 1.15 mmol) was reacted with tetrahydrothiopyrane (0.25 mL, 2.37 mmol) and AgBF4 (0.46 g, 2.37 mmol) to form A8 (0.42 g, 1.03 mmol) as yellowish oil, yield 65%, Rf = 0.32 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.92 (t, J = 5.4 Hz, 2 H, CH2), 3.64-3.63 (m, 2 H, CH2), 3.59-3.54 (m, 10 H, 5 CH2), 3.62-3.49 (m, 4 H, 2 CH2), 3.41 (t, J = 6.6 Hz, 2 H, CH2), 3.23 (m, 2 H, CH2), 2.16-2.10 (m, 2 H, CH2), 1.90-1.82 (m, 2 H, CH2), 1.77-1.70 (m, 1 H, CH2), 1.66-1.59 (m, 1 H, CH2), 1.53 (q, J = 6.9 Hz, 2 H, CH2), 1.28 (m, 12 H, 6 CH2), 0.89 (t, J = 6.9 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.39, 70.80, 70.67, 70.60 (2 C), 70.27, 70.23, 65.47, 40.56, 37.33 (2 C), 32.19, 29.98, 29.89, 29.78, 29.60, 26.45, 22.96, 22.90, 21.46 (2 C), 13.98, HR-MS (ESI) m/z: Calcd for C22H45O4S {[M-Cl-]+} 405.3033, found: 405.3246.
S-(2-(2-2-(2-(dodecyloxy)ethoxy)ethoxy)ethoxy)ethyl-tetrahydro-thiophenium chloride(A9)
In the general procedure for preparing A, Q5 (0.81 g, 1.72 mmol) was reacted with tetrahydrothiophene (0.23 mL, 2.58 mmol) and AgBF4 (0.51 g, 2.58 mmol) to form A9 (0.46 g, 1.07 mmol) as yellowish oil, yield 62%, Rf = 0.33 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.89 (t, J = 5.4 Hz, 2 H, CH2), 3.64 (t, J = 4.5 Hz, 2 H, CH2) (m, 2 H, 2 CH2), 3.59-3.59 (m, 10 H, 5 CH2), 3.47-3.39 (m, 4 H, 2 CH2), 3.36 (t, J = 6.6 Hz, 2 H, CH2), 2.33-2.19 (m, 4 H, 2 SCH2CH2), 1.52 (q, J = 6.6 Hz, 2 H, CH2), 1.32-1.28 (m, 18 H, 9 CH2), 0.88 (t, J = 6.9 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.26, 70.68, 70.91, 70.65, 70.64, 70.35, 70.27, 65.89, 44.38 (2 C), 43.38, 32.23, 30.01, 29.98, 29.95 (3 C), 29.79, 29.67, 28.81 (2 C), 26.47, 22.98, 14.01, HR-MS (ESI) m/z: Calcd for C24H49O4S {[M-Cl-]+} 433.3346, found: 433.3700.
S-(2-(2-2-(2-(dodecyloxy)ethoxy)ethoxy)ethoxy)ethyl-tetrahydro-thiopyranium chloride (A10)
In the general procedure for preparing A, Q5 (0.75 g, 1.59 mmol) was reacted with tetrahydrothiopyrane (0.25 mL, 2.37 mmol) and AgBF4 (0.46 g, 2.37 mmol) to form A10 (0.40 g, 0.89 mmol) as yellowish oil, yield 56%, Rf = 0.34 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.94 (t, J = 5.4 Hz, 2 H, CH2), 3.66-3.65 (m, 2 H, CH2), 3.61-3.56 (m, 8 H, 4 CH2), 3.53-3.47 (m, 6 H, 3 CH2), 3.42 (t, J = 6.9 Hz, 2 H, CH2), 3.25-3.20 (m, 2 H, CH2), 2.18-2.12 (m, 2 H, CH2), 1.92-1.85 (m, 2 H, CH2), 1.77-1.73 (m, 1 H, CH2), 1.66-1.63 (m, 1 H, CH2), 1.54 (q, J = 6.9 Hz, 2 H, CH2), 1.34-1.26 (m, 18 H, 9 CH2), 0.91 (t, J = 6.9 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.54, 70.95, 70.83, 70.75 (2 C), 70.43, 70.39, 65.59, 40.70, 37.49, 36.40, 32.36, 30.14, 30.10, 30.07 (4 C), 29.92, 29.80, 26.60, 23.11, 23.06, 21.58, 14.12, HR-MS (ESI) m/z: Calcd for C25H51O4S {[M-Cl-]+} 447.3503, found: 447.3604.
S-(2-(2-2-(2-(hexadecyloxy)ethoxy)ethoxy)ethoxy)ethyl-tetrahydro-thiophenium chloride (A11)
In the general procedure for preparing A, Q6 (0.64 g, 1.21 mmol) was reacted with tetrahydrothiophene (0.16 mL, 1.82 mmol) and AgBF4 (0.35 g, 1.82 mmol) to form A11 (0.30 g, 0.62 mmol) as yellowish oil, yield 51%, Rf = 0.35 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.90 (t, J = 5.4 Hz, 2 H, CH2), 3.65-3.63 (m, 2 H, CH2), 3.59-3.58 (m, 2 H, CH2), 3.55-3.52 (m, 6 H, 3 CH2), 3.51-3.59 (m, 4 H, 2 CH2), 3.48-3.44 (m, 2 H, CH2), 3.41-3.38 (m, 4 H, 2 CH2), 2.31-2.27 (m, 2 H, SCH2CH2), 2.24-2.21 (m, 2 H, SCH2CH2), 1.52 (q, J = 6.9 Hz, 2 H, CH2), 1.27 (m, 26 H, 13 CH2), 0.88 (t, J = 6.9 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.36, 70.93, 70.69, 70.67, 70.65, 70.35, 70.28, 65.90, 44.54 (2 C), 43.41, 32.20, 30.01, 29.95, 29.94 (3 C), 29.92, 29.91 (2 C), 29.90, 29.77, 29.64, 28.84 (2 C), 26.45, 22.96, 13.97, HR-MS (ESI) m/z: Calcd for C28H57O4S {[M-Cl-]+} 489.3972, found: 489.4201.
S-(2-(2-2-(2-(hexadecyloxy)ethoxy)ethoxy)ethoxy)ethyl-tetrahydro-thiopyranium chloride (A12).
In the general procedure for preparing A, Q6 (0.60 g, 1.14 mmol) was reacted with tetrahydrothiopyrane (0.18 mL, 1.71 mmol) and AgBF4 (0.33 g, 1.71 mmol) to form A12 (0.28 g, 0.55 mmol) as yellowish oil, yield 48%, Rf = 0.33 (methanol-dichloromethane 1:9). 1H NMR (CD3CN, δ): 3.91 (t, J = 5.4 Hz, 2 H, CH2), 3.64-3.62 (m, 2 H, CH2), 3.58-3.57 (m, 2 H, CH2), 3.55-3.53 (m, 6 H, 3 CH2), 3.51-3.46 (m, 4 H, 2CH2), 3.40 (t, J = 6.0 Hz, 2 H, CH2), 3.22 (m, 2 H, CH2), 2.17-2.10 (m, 4 H, 2 CH2), 1.90-1.82 (m, 2 H, CH2), 1.75-1.69 (m, 1 H, CH2), 1.66-1.68 (m, 1 H, CH2), 1.54-1.50 (q, J = 6.0 Hz, 2 H, CH2), 1.27 (m, 26 H, 13 CH2), 0.88 (t, J = 6.0 Hz, 3 H, CH3), 13C NMR (CD3CN, δ): 71.36, 70.87, 70.75, 70.71, 70.68, 70.35, 70.31, 65.48, 40.58, 37.35 (2 C), 32.21, 30.06, 29.95 (4 C), 29.93, 29.77, 29.65, 29.91, 29.77, 29.65, 26.48, 22.97, 22.91, 21.43 (2 C), 13.97, HR-MS (ESI) m/z: Calcd for C29H59O4S {[M-Cl-]+} 503.4129, found: 503.4427.
2. Preparation of the SL/DNA condensate and agarose gel retardation
The amount of SL used in forming SL/DNA nano particles was calculated through the sulfonium amount over phosphate of DNA (S/P ratio). SLs stock solution was prepared by dissolving SLs in acetonitrile.
The condensation ability of each SL to bind pDNA at different S/P ratios was examined by means of agarose gel electrophoresis. To set up the gel electrophoresis experiment, appropriate volumes of SLs stock were diluted in HEPE buffer (4-(2-hydroxyerhyl)piperazine-1-erhaesulfonic acid, pH = 7.5,5 mmol/L) to form a group of SL samples with total volume 5 μL. SL/DNA nanoparticles were formed by adding 3 μL aqueous eGFP plasmid solution (600 ng) dropwisely to SL samples. The mixtures were shaken gently for 10 s, and then cultured in 37℃ for 30 min. SL/DNA particles were formed at S/P ratios 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, respectively.
The condensate solution (8 μL) mixed with loading buffer containing Bromophenol blue (2 μL) was added to the agarose gel. The samples were electrophoresed on the 1% (w/v) agarose gel containing Ethidium bromide (EB) and Tris-acetate (TAE) running buffer at 135 V for 30 min in a Sub-Cell system. The electrophoresis results were visualized with a UV lamp using a Bio-Rad Universal Hood II.
3. SL/DNA condensate particle size and zeta potential measurements
The particle size and zeta potential of the condensates were determined through Nano-ZS 90 Laser Particle Size and Zeta Potential Analyzer
A series volumes of SLs acetonitrile stock solutions were diluted with HEPE (pH = 7.5,5 mmol/L) to a total volume 28 μL. Each of the solutions was added drop wisely to aqueous eGFP plasmid solution (28 μL, 800 ng) to form SL/DNA particles at S/P ratios 10:1, 20:1, 30:1, 40:1, and 50:1, respectively. The mixtures were shaken gently for 10 s, and then cultured in 37℃ for 30 min. The SL/DNA condensate solutions were diluted with water to 1 mL and measured partial size and Zeta potential using Nano-ZS 90 Laser Particle Size and Zeta Potential Analyzer. The mean diameter and population distribution were computed from the diffusion coefficient using a multimodal cumulate analysis supplied by the manufacturer.
4. SL cytotoxicity
The cytotoxicities of SLs were tested on HepG 2 cell lines by MTT assay. HepG 2 cells were cultured in DMEM media supplemented with FBS (10%, v/v) and streptomycin (100 μg/mL) in a humidified 5% CO 2 incubator at 37°C.
The cells were seeded in 96-well plates at 8000 cells and 200 μL medium per well, and cultured for 8 h to allow attachment. The medium was removed and 200 μL DMEM medium with 1% FBS was added to wells. The cells were incubated for 2 h, then were treated with 1μL DMSO solution of different concentrations and different concentrations (9.38 mg/mL, 4.69 mg/mL, 2.34 mg/mL, 0.29 mg/mL, 0.15 mg/mL, 0.01 mg/mL) of SLs. PEI solutions were used as positive control. Cells were further incubated for 24 h and 48 h. After that, the medium was removed, 90 μL DMEM medium without FBS was added, and then 10 μL of MTT (5 mg mL −1) was added to wells, and the cells were incubated for another 4 h. Finally, MTT was replaced with 150 µL of DMSO, the plates were oscillated for 10~20 min. The absorbance of the purple formazan was recorded at 490 nm using a Thermo Scientific Multiskan GO. The relative viability of the cells was calculated based on the data of five parallel tests by comparing them to the controls.
6. Cellular Uptake
The eGFP plasmid was labeled with Cy5 red dye to Label CY5 Labeling Kit according to the manufacturer’s protocol. The cellular uptakes of SL/Cy5-DNA complexes into HepG2 cells were observed by Fluorescence microscope. 25 kDa PEI was used as control.
SL/Cy5-DNA condensates were prepared at S/P ratio of 10/1 (PEI/ Cy5-DNA was at N/P = 10/1). The stock SL acetonitrile solutions were diluted with serum free medium to a volume of 10 uL. The condensates were formed by adding diluted SL solution to DNA solution (200 ng, 10 μL serum-free culture medium) and stood still at 37 °C for 30 min.
HepG2 cells were seeded into 96-well plates (5000 cells/well) and allowed to attach and grow for 24 h until the cells reached 70-80% confluence. The medium was replaced with serum-free culture medium (100 μL). The HepG2 cells were added SL/Cy5-DNA nanoparticles and incubated for 4 h at 37 ◦C. Subsequently, the cells were washed with 3 ×PBS and fixed with 4 % paraformaldehyde for 20 min. Following removal of paraformaldehyde, the cells were washed three times with PBS and sealed in tablet with DAPI stain. The fluorescence of the cells was visualized with a Leica biological microscope DMI 4000B at excitation and emission wavelengths of 649/670 nm for Cy5 (red) and 364/454 nm for DAPI (blue), respectively.