Reducible amino acid based cationic lipids with a naphthalimide moiety as non-viral gene vehicles

Abstract

Three basic amino acid–based cationic lipids bearing a fluorescent naphthalimide moiety and a reducible disulfide linkage are synthesized and applied as non-viral gene vehicles. Their DNA interactions are investigated by agarose-gel retardant and ethidium bromide replacement assays. The sizes and zeta potentials of the liposome/DNA complexes are measured by dynamic light scattering. The cytotoxicities of the liposome/DNA complexes are examined using HeLa and 7702 cell lines by MTT assays. The glutathione-responsive DNA release process is studied through time-dependent fluorescence assays. Luciferase gene expression showed the transfection efficiency of the liposome is dramatically increased in the presence of 10% serum. Confocal laser scanning microscopy studies corroborated that the liposome/DNA complexes are successfully uptaken into HeLa cells. These results demonstrate the promising use of amino acids and naphthalimide-containing lipids for safe and efficient gene delivery.

Reducible amino acid based cationic lipids with a naphthalimide moiety were synthesized and applied as non-viral gene vehicles

Introduction

Gene therapy offers a promising strategy to prevent or cure human diseases of genetic origin by introducing exogenous genetic materials, such as DNA, RNA, or small interfering RNA (siRNA) into cells. ¹ However, its clinical use is impeded by a lack of safe and effective delivery carriers. ² As an important type of non-viral gene vehicles, cationic lipids have been widely investigated for their facile synthesis, low toxicity, and excellent biocompatibility compared to viral vectors.^3,4 The structures of cationic lipids are characterized by three components: a hydrophilic head group, a hydrophobic tail, and a linker between the former two moieties.^5–7 All these components can seriously affect the toxicity and transfection efficacy of the final lipids.

Gene transfection with cationic lipids needs to satisfy the contrary requirements of adequate extracellular DNA protection and efficient intracellular DNA release. ⁸ Amino acids and synthetic peptides with desirable physicochemical properties and good biocompatibility are prospective substances for the construction of the multivalent polar domains which can complex negatively charged nucleic acids via strong electrostatic interactions, leading to improved DNA condensation and enhanced cellular uptake.^9–11 In our previous studies, several amino acid–based amphiphiles with multicharge character were found to have the ability to achieve efficient gene delivery with low cytotoxicity.^11–15 To facilitate DNA release in the cell, gene delivery vectors with stimuli-responsive ability have attracted increasing interest. ¹⁶ Among diverse stimuli, the significant difference in the concentration of the reducing tripeptide glutathione (GSH) between extracellular and intracellular environments makes disulfide bonds unique in biology and in gene delivery systems. ⁸ In a characteristic tether group, disulfide can be degraded in the cytoplasm and facilitates the dissociation of vectors and DNA by specifically responding to the redox potential through thiol–disulfide exchange reactions.^17,18 Many disulfide-bridged nanocarriers with high transfection efficiency (TE) and low toxicity have been synthesized.^{11,12,19–22} Aliphatic chain, cholesterol, and even tocopherol derivatives are commonly used as hydrophobic components to construct cationic lipids. ⁵ The design and synthesis of new lipid systems with alternative structural types are crucial for the development of potent synthetic gene vectors. ¹⁴ The rigid naphthalimide moiety with native hydrophobic properties can be used to functionalize cationic lipids.^4,23–26 Moreover, it has high DNA complexing ability, strong yellow-green fluorescence, and good photostability. ²⁷ Naphthalimide factionalized lipids with good fluorescence properties have been used to monitor the process of cellular uptake, DNA translocation, and release.^4,23–26 Although a series of naphthalimide-modified lipids has been synthesized, naphthalimide-containing cationic lipid gene vehicles with reduction-responsive ability have been seldom studied.

Based on the above considerations, herein we have designed and synthesized three lipidic gene vectors with a multivalent amino acid head group, a reduction-responsive disulfide linker, and a rigid naphthalimide tail (Scheme 1). Their DNA interaction, physicochemical properties, cytotoxicities, gene transfection activity as well as cellular uptake have been systematically investigated.

Scheme 1.

Synthesis of lipids 5a–c. Reagents and conditions: (a) Et₃N, (b) CF₃COOH, CH₂Cl₂, and (c) N^α-Boc-l-histidine, N,N^ω,N^ω’-Tris-Boc-l-arginine, or N^α,N^ε-di-Boc-l-lysine, EDC·HCl, HOBt, and DIEA.

Results and discussions

Syntheses of new amino-based cationic lipids

Lipids 5 were synthesized as shown in Scheme 1. Compound 3 was prepared by coupling compound 1 and Boc-protected cystamine 2 (Boc = tert-butyloxycarbonyl). Subsequently, precursors 4a–c were obtained by coupling the deprotection product of 3 with Boc-protected amino acids (N^α,N^β-di-Boc-l-lysine, N^α,N^ω,N^ω’-Tris-Boc-l-arginine, or N^α-Boc-l-histidine) in the presence of EDC·HCl and HOBt. Finally, the target lipids were obtained by removing the Boc groups with trifluoroacetic acid in anhydrous CH₂Cl₂. The structures of the lipidic compounds were characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS).

UV-absorption and fluorescence of lipid 5c

The photophysical properties of lipid 5c were examined in deionized water, and the results are shown in Figure 1. Lipid 5c displayed a broad intramolecular charge transfer (ICT) absorption band centered at 400 nm (ε = 14,760 M⁻¹ cm⁻¹) and high-energy π–π* transitions at ca. 250 nm (10,280 M⁻¹ cm⁻¹) (Figure 1(a)). Excitation of 5c at 445 nm resulted in a broad emission band centered at 550 nm (Figure 1(b)).

Figure 1.

(a) UV-Vis absorption spectra of 5c at various concentrations in deionized water. Inset: the plot of concentration of 5c versus absorbance at 450 or 250 nm. (b) The molar absorption coefficient of 5c and its emission spectrum in deionized water.

Preparation and characterization of liposomes and liposome/DNA complexes (lipoplexes)

The cationic liposomes were prepared by the thin-film hydration method and a lipid/DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) ratio of 1:1 was used herein. To evaluate their DNA-binding abilities, both the agarose-gel retardant and ethidium bromide (EB) replacement assays were employed.^12,18 The agarose-gel retardant assay shows that the liposomes formed from 5b,c had higher DNA-binding ability than that formed from 5a, with complete DNA retardation achieved at an N/P ratio (the amino groups of liposome to phosphate groups of DNA) of 2 for liposomes 5b,c and 4 for liposome 5a (Figure 2(a)). The reason may due to the weak basic character of the imidazole moiety.^12,28,29 In addition, the EB exclusion assay (Figure 2(b)) shows that liposome 5a with (l)-histidine as the cationic headgroup exhibited a weaker fluorescence quenching effect. This indicated that liposome 5a had a lower pDNA-binding affinity than those of 5b,c. The results correlated well with the gel retardation assay (Figure 2(a)).

Figure 2.

Electrophoretic gel retardation assays (a) and ethidium bromide displacement assays (b) of pDNA binding for liposomes 5a–c/DOPE/pDNA complexes under various N/P ratios.

To gain an insight into whether the disulfide linker is reducible by reducing molecules such as GSH, fluorescence assays were used to monitor the time-dependent degradation of liposomes 5b in the presence of 10 mM GSH. On increasing the incubation time, the fluorescence intensity of naphthalimide-fuctionlized liposome 5b decreased gradually (Figure 3). This demonstrated that GSH enabled cleavage of the disulfide in amino acid–based delivery carriers.

Figure 3.

Fluorescence assays of liposomes 5b (1 mM) in the presence of 10 mM GSH with different incubation times.

Sizes and zeta potentials of the formed lipoplexes

The particle sizes of different lipoplexes were measured by dynamic light scattering (DLS) assays, and the results are shown in Figure 4(a). Liposomes 5a–c could condense DNA into lipoplex nanoparticles with diameters in the range of 170–320 nm, which strongly depended on the N/P ratio. The particle size reaches its maximum when N/P is 1. Further increasing the N/P ratio to 8 resulted in smaller particles (200 nm).

Figure 4.

Mean particle sizes (a) and surface charges (b) of the lipoplexes at various N/P ratios. Data represent mean value ± SD (n = 3).

The surface potentials of the lipoplex nanoparticles were also measured by DLS, and the results are shown in Figure 4(b). The zeta potentials of all the lipoplexes showed a similar trend on changing the N/P ratio: they increased from −38 to 36 mV when the N/P ratio increased from 1 to 16. The zeta potentials increased and turned positive with an increase in cationic materials.

Cytotoxicity

The cytotoxicities of the prepared lipoplexes were examined using the HeLa (human cervical cancer epithelia) and 7702 (normal human Liver cell lines) cell lines in serum-free medium by employing the MTT assay, with lipofectamine 2000 and PEI 25K as the control. The results shown in Figure 5 revealed that the chemical structure of the cationic headgroup significantly affected the cytotoxicity of the lipoplexes. Both HeLa and 7702 cells remained at a survival rate of over 85% for lipoplexes 5a,b even when the N/P ratio reached 12. However, HeLa cells were more sensitive to lipoplexes 5c, with the percentage of viable cells decreasing rapidly on increasing with N/P ratio. On the contrary, the 7702 live cells were less sensitive.

Figure 5.

Cell toxicities of the lipoplexes 5 prepared at various N/P ratios in serum-free medium with lipofectamine 2000 and PEI 25K (N/P = 10) as controls. HeLa cells (a) and 7702 cells (b). Data represent mean value ± SD (n = 3).

In vitro transfection

The in vitro gene transfection efficiencies of the liposomes were studied with HeLa and 7702 cells using the pGL3 plasmid as a luciferase reporter gene. Figure 6a-b exhibit TEs at different N/P ratios in serum-free medium; lipofectamine 2000 and PEI 25K were used as controls. For both cell lines, liposomes 5b,c gave higher TE values than 5a under all the N/P ratios tested. We speculate that the higher DNA-binding ability of liposomes 5b,c would benefit their transfection behaviors. However, their TE values were lower than those of lipofectamine 2000 and PEI 25K.

Figure 6.

Luciferase expression induced by liposomes 5 at various N/P ratios in HeLa cells (a) and 7702 cells (b) in serum-free medium with PEI 25K (N/P = 10) as the control. Data represent mean value ± SD (n = 3).

It was found that the serum inhibits the transfection of cationic vectors. ³⁰ Due to the better transfection performance of liposomes 5b,c in HeLa and 7702 cells, transfection with these liposomes was studied in the same cell lines in the presence of 10% fetal bovine serum (FBS) and with PEI 25K as the control (Figure 7). The TEs of 5b,c at N/P = 12 in the presence of 10% FBS were dramatically increased twofold as compared with the TEs of 5b,c in serum-free medium in HeLa cells (Figure 7(a)). In addition, the optimum TE of liposome 5b (N/P = 12) in FBS-containing medium was almost the same as that in the serum-free medium in 7702 cells. Interestingly, the optimum TE of 5c (N/P = 12) was dramatically increased threefold as compared with the TE of 5c (N/P = 10) in the absence of serum in the same cell line (Figure 7(b)). In contrast, the TE of PEI-25K decreased sharply in both cell lines (Figure 7(a) and (b)). Further mechanistic research is now in progress.

Figure 7.

Luciferase expression induced by liposomes 5 at various N/P ratios in HeLa cells (a) and 7702 cells (b) in the presence of 10% FBS with PEI 25K (w/w = 1.4) as the control. Data represent mean value ± SD (n = 3). Paired t-test for 5c and two independent t-tests for PEI 25K.

Cellular uptake of the amino acid–based cationic liposomes

Cellular uptake studies were performed in HeLa cells using Cy5-labeled dsDNA. Cy5-labeled DNA emitted red fluorescence under fluorescence microscopy conditions, while liposomes 5c gave rise to a strong green fluorescence. To locate the liposome 5c/DNA lipoplexes, the nuclei of the cells were stained with a nucleus-specific blue fluorescent dye (DAPI). As shown in Figure 8, after incubation for 2 h with the liposome 5c/Cy5-pDNA lipoplex (N/P = 6 and 8), almost all the cells were stained by liposome 5c (green) and Cy5-labeled dsDNA (red), which clearly indicates that the cellular uptake of the lipoplexes was successful.

Figure 8.

Confocal laser scanning microscopy (CLSM) images of HeLa cells transfected with Cy5-labeled DNA by liposome 5c at N/P ratio of 6 (a) and 8 (b) (red: Cy5-labeled pDNA; green: liposome 5c; blue: DAPI-stained cell nuclei).

Conclusion

Three amino acid–based amphiphilic molecules bearing a naphthalimide moiety and a biodegradable disulfide linkage have been designed and synthesized. The liposomes formed from these lipids can condense stable lipoplex nanoparticles and deliver pDNA into cells. The structure of the hydrophilic headgroup significantly affected the TE values and cytotoxicities. Arginine- and lysine-containing lipids 5b,c with higher DNA-binding ability gave higher TE than the histidine derivative. Although the TEs of 5 were still lower than that of the commercial transaction agent PEI 25K, these lipoplexes showed good serum-resistance capability. Further studies to increase the TE value and to elucidate the intracellular trafficking mechanisms of this type of cationic lipids are now in progress.

Experimental section

Materials

N^α,N^β-di-Boc-l-lysine, N^α-Boc-l-histidine and N^α,N^ω,N^’-Tris-Boc-l-arginine were purchased from GL Biochem (Shanghai, China). DOPE was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Compounds 1 ³¹ and 2 ³² were synthesized following the procedures reported in the literature. Other materials and instruments used herein were the same as those mentioned in the literature.^10–12 The experimental methods for the EB displacement assay, cell cultures, in vitro transfection, and cytotoxicity studies were performed according to the literature procedures.^11–13

Synthesis of lipids 5a–c

Preparation of compound 3

Triethylamine (101.2 mg, 1.0 mmol) was added drop wise to a solution of compound 1 (375.3 mg, 1 mmol) and 2 (1.262 g, 5.0 mmol) in DMSO (30 mL), and the resulting mixture was stirred at 110 °C for 15 h. The mixture was then diluted with ethyl acetate (200 mL), followed by washing with water (100 mL) and brine (100 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated to afford a yellow solid, which was further purified by column chromatography on silica gel (PE/EtOAc = 2:1, v/v) to yield 3 as a light-yellow solid (210 mg, 38%). ¹H NMR (400 MHz, CDCl₃): δ 8.57 (d, J = 7.2 Hz, 1H), 8.45 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 4.13 (t, J = 8.0 Hz, 2H), 3.79 (t, J = 6.0 Hz, 2H), 3.49 (d, J = 6.0 Hz, 2H), 3.09 (t, J = 6 Hz, 2H), 2.83 (t, J = 6.0 Hz, 2H), 1.74-1.66 (m, 2H), 1.43 (s, 9H), 1.37-1.19 (m, 10H), 0.85 (t, J = 6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 164.60, 164.05, 155.90, 148.96, 134.22, 131.12, 126.45, 124.82, 123.00, 120.48, 110.69, 104.15, 79.78, 42.13, 40.24, 39.44, 38.03, 37.08, 31.81, 29.39, 29.24, 28.37, 28.22, 27.19, 22.64, 14.09. HRMS (ESI): [M + H]⁺ calcd for C₂₉H₄₂N₃O₄S₂: 560.2611; found: 560.2606.

Preparation of compounds 4

Compound 3 (1 mmol) was dissolved in anhydrous CH₂Cl₂ (2 mL), then CF₃COOH (2 mL) was added at 0 °C. After stirring for 6 h, the solvent was removed under reduced pressure. The residue was dissolved in DMF (20 mL), then N^α-Boc-l-histidine (225 mg, 1 mmol), N^α,N^ω,N^ω’-Tris-Boc-l-arginine (475 mg, 1 mmol) or N^α,N^β-di-Boc-l-lysine (346 mg, 1 mmol), 1-hydroxybenzotriazole (HOBt, 162 mg, 1.2 mmol), EDC·HCl (230 mg, 1.2 mmol), and N,N-diisopropylethylamine (DIEA, 202 mg, 2 mmol) were added, and the reaction mixture was stirred for 1 h at 0 °C, and then at room temperature overnight. The mixture was washed with saturated aqueous NaHCO₃ solution (2 × 20 mL) and brine (20 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated to afford a white solid, which was further purified by column chromatography on silica gel to yield 4 as a yellow solid.

Compound 4a yield 44%

¹H NMR (400 MHz, DMSO): δ 8.63 (d, J = 8.3 Hz, 1H), 8.39 (d, J = 7.1 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.97 (s, 1H), 7.89 (s, 1H), 7.51 (s, 1H), 6.72 (s, 1H), 4.10-4.06 (m, 1H), 3.96 (t, J = 8.0 Hz, 2H), 3.67 (d, J = 5.9 Hz, 2H), 3.03 (t, J = 6.6 Hz, 2H), 2.85-2.66 (m, 4H), 2.47 (s, 4H), 1.56 (s, 2H), 1.34-1.14 (m, 19H), 0.80 (d, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, DMSO): δ 172.10, 164.08, 163.28, 155.53, 150.50, 135.09, 134.53, 131.05, 129.73, 128.84, 124.79, 122.30, 120.56, 108.59, 104.22, 78.52, 54.94, 42.50, 38.43, 37.44, 36.35, 31.66, 30.05, 29.17, 29.03, 28.57, 28.03, 27.00, 22.50, 14.36. HRMS (ESI): [M + H]⁺ calcd for C₃₅H₄₈N₆O₅S₂: 697.3200; found: 697.3206.

Compound 4b yield 41%

¹H NMR (400 MHz, DMSO-d₆): δ 8.62 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 7.1 Hz, 1H), 8.00 (t, J = 6.0 Hz, 1H), 7.83 (s, 1H), 6.82 (d, J = 6.0 Hz, 1H), 3.98-3.90 (m, 2H), 3.88-3.86 (m, 1H), 3.72-3.67 (m, 2H), 3.21 (d, J = 5.2 Hz, 2H), 3.03 (t, J = 6.6 Hz, 2H), 2.86 (s, 2H), 2.77 (t, J = 6.6 Hz, 2H), 1.61-1.12 (m, 39H), 0.80 (t, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, DMSO): δ 172.51, 164.11, 163.53, 163.31, 162.74, 155.66, 152.47, 150.52, 134.57, 131.11, 129.76, 128.83, 124.85, 122.34, 120.59, 108.62, 104.26, 83.24, 78.51, 54.43, 42.50, 38.32, 37.56, 36.29, 31.68, 31.20, 29.08, 28.50, 28.01, 26.97, 22.49, 14.35. HRMS (ESI): [M + H]⁺ calcd for C₄₅H₆₉N₇O₉S₂: 916.4171; found: 916.4677.

Compound 4c yield 46%

¹H NMR (400 MHz, DMSO): δ 8.58 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 7.3 Hz, 1H), 8.19 (d, J = 8.5 Hz, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 3.93 (t, J = 8.0 Hz, 2H), 3.83-3.78 (m, 1H), 3.68-3.63 (m, 2H), 3.03 (t, J = 8.0 Hz, 2H), 2.88-2.75 (m, 4H), 1.53-1.17 (m, 36H), 0.78 (t, J = 8.0 Hz, 3H). ¹³C NMR (100 MHz, DMSO): δ 172.78, 164.06, 163.26, 155.98, 155.72, 150.46, 134.50, 131.03, 129.71, 128.77, 124.75, 122.29, 120.55, 108.61, 104.18, 78.37, 77.72, 54.79, 42.51, 37.56, 36.36, 32.11, 31.66, 29.64, 29.10, 28.64, 28.03, 27.00, 23.25, 22.49, 14.34. HRMS (ESI): [M + Na]⁺ calcd for C₄₀H₆₁N₅O₇S₂ 810.3905; found: 810.3912.

Preparation of lipids 5

Compounds 4 (0.25 mmol) was dissolved in anhydrous CH₂Cl₂ (2 mL), and then CF₃COOH (2 mL) was added at 0 °C. After stirring for 6 h, the solvent was removed under reduced pressure, and the yellow product was washed twice with ether to afford the title lipids 5 as yellow oils.

Lipid 5a yield 93%

¹H NMR (400 MHz, DMSO): δ 8.62 (d, J = 8.3 Hz, 1H), 8.38 (d, J = 7.1 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.66 (t, J = 8.0 Hz, 2H), 7.50 (s, 1H), 6.75 (s, 1H), 4.09-4.05 (m, 1H), 3.96 (t, J = 8.0 Hz, 2H), 3.68-3.66 (m, 2H), 3.02 (t, J = 6 Hz, 2H), 2.85-2.70 (m, 4H), 2.45 (s, 4H), 1.57-1.54 (m, 2H), 1.25-1.20 (m, 10H), 0.79-0.81 (m, 3H). ¹³C NMR (100 MHz, DMSO): δ 172.10, 164.01, 163.24, 150.45, 135.12, 134.48, 131.01, 129.78, 128.86, 124.67, 122.30, 120.53, 108.52, 104.27, 54.94, 42.46, 38.46, 37.44, 36.35, 31.68, 30.02, 29.17, 29.03, 28.01, 27.00, 22.48, 14.10. HRMS (ESI): [M + H]⁺ calcd for C₃₀H₄₀N₆O₃S₂: 597.2676; found: 597.2680.

Lipid 5b yield 88%

¹H NMR (400 MHz, DMSO-d₆): δ 8.66 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 7.1 Hz, 1H), 8.02 (t, J = 6.0 Hz, 1H), 7.91 (s, 2H), 7.69 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 4.00-3.97 (m, 2H), 3.88-3.86 (m, 1H), 3.72-3.65 (m, 2H), 3.22 (d, J = 5.2 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.86 (s, 4H), 2.77 (t, J = 6.6 Hz, 2H), 1.60-1.21 (m, 14H), 0.85 (t, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, DMSO): δ 172.50, 164.08, 163.48, 150.45, 134.62, 131.10, 129.78, 128.81, 124.88, 122.32, 120.58, 108.62, 104.28, 54.43, 42.52, 38.30, 37.62, 36.42, 36.24, 31.70, 31.60, 31.20, 29.20, 29.08, 28.01, 26.97, 22.47, 14.20. HRMS (ESI): [M + H]⁺ calcd for C₃₀H₄₅N₇O₃S₂: 616.3098; found: 616.3095.

Lipid 5c yield 94%

¹H NMR (400 MHz, DMSO): δ 8.57 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 7.3 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 3.93 (t, J = 8.0 Hz, 2H), 3.83-3.79 (m, 1H), 3.67-3.62 (m, 2H), 3.03 (t, J = 8.0 Hz, 2H), 2.84-2.76 (m, 4H), 1.50-1.15 (m, 18H), 0.80 (t, J = 8.0 Hz, 3H). ¹³C NMR (100 MHz, DMSO): δ 172.60, 164.10, 163.32, 150.46, 134.52, 131.03, 129.74, 128.79, 124.78, 122.26, 120.55, 108.63, 104.18, 54.75, 42.51, 38.35, 37.62, 36.36, 32.13, 31.68, 29.64, 29.20, 29.03, 28.02, 27.00, 23.25, 22.53, 14.32. HRMS (ESI): [M + Na]⁺ calcd for C₃₀H₄₅N₅O₃S₂: 610.2856; found: 610.2851.

Preparation of the cationic liposomes

The cationic lipid (0.0025 mmol) and neutral colipid (DOPE, 0.0025 mmol) were dissolved in chloroform (2.5 mL) in autoclaved glass vials. The solvent was removed with a thin flow of moisture-free nitrogen gas, and the dried lipid film was then kept under high vacuum for 8 h. A portion of 2.5 mL of sterile deionized water was added to the vacuum-dried lipid film, and the mixture was allowed to swell overnight. The liposomes were vortexed for 1−2 min to remove any adhering lipid film and sonicated in a bath sonicator for 5 min at room temperature to produce multilamellar vesicles (MLVs). The MLVs were then sonicated in an ice bath using a probe sonifier to afford the corresponding cationic liposomes (1.0 mmol L⁻¹).^11–13

Agarose-gel retardation assays

Lipids 5/DOPE/pDNA complexes at different N/P ratios (the amino groups of the lipids (N) to the phosphate groups (P) of DNA) ranging from 0.5 to 8 were prepared by adding an appropriate volume of liposome to pUC-19 DNA (0.125 μg). The complexes were incubated at 37 °C for 30 min. Thereafter, the complexes were electrophoresed on 1.0% (w/v) agarose-gel containing GelRed and with Tris-acetate (TAE) running buffer at 110 V for 30 min. DNA was visualized with a UV lamp at a wavelength of 312 nm using a BioRad Universal Hood II (BioRad, USA).^11–13

EB replacement assays

The ability of liposomes 5 to condense DNA was studied using EB exclusion assays. Fluorescence spectra were measured at room temperature in air on a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer (Horiba, USA) and corrected for the system response. EB (5 μL, 1.0 mg mL⁻¹) was placed in a quartz cuvette containing 2.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) solution (pH = 7.4). After shaking, the fluorescence intensity of EB was measured. Then, CT DNA (10 μL, 1.0 mg mL⁻¹) was added to the solution with mixing. The measured fluorescence intensity is the result of the interaction between DNA and EB. Subsequently, the solution of liposomes 5 (1.0 mM, 7.5 μL for each addition) was added to the above solution for further measurement. All the samples were excited at 520 nm, and the emission was measured at 600 nm. The pure EB solution and DNA/EB solution without cationic liposomes were used as negative and positive controls, respectively. The percent relative fluorescence (%F) was determined using the equation %F = (F − FEB)/(F0 − FEB), wherein FEB and F0 denote the fluorescence intensities of pure EB solution and DNA/EB solution, respectively.^11–13

Fluorescence assays

The fluorescence change of the liposomes in response to GSH was examined using fluorescence assays. The liposome solution (2.0 mM) in deionized water (250 μL) was added with GSH (250 μL, 20 mM). After incubation of the solution at 37 °C for different times (3, 8, 12 and 30 h), 30 μL of the above solution was diluted with deionized water (3 mL), and then the fluorescence spectra were obtained. Excitation wavelength at 445 nm, emission band centered at 550 nm.

Lipoplex particle sizes and zeta potentials

The sizes and zeta potentials of the liposome/pDNA lipoplexes at various N/P ratios were analyzed at room temperature using a DLS system (Zetasizer Nano ZS, Malvern Instruments Led, United Kingdom at 25 °C. The lipoplex particle solutions were first prepared by mixing 5/DOPE liposomes and pDNA (1 μg mL⁻¹) under different N/P charge ratios in deionized water (1 mL).^11–13

MTT cytotoxicity assays

The toxicities of the liposomes and lipoplexes toward 7702 and HeLa cells were determined using the MTT assay following literature procedures. ¹¹ About 7000 cells/well were seeded into 96-well plates. For lipoplexes, after 24 h, optimized lipid/DOPE formulations were complexed with 0.2 μg of DNA at various N/P ratios. Lipoplexes prepared from lipofectamine 2000 and polyplexes prepared from PEI-25K were used as controls.^11–13

In vitro transfection assays

7702 and HeLa cells were seeded into 24-well plates at densities of 80,000 cells/well in 0.5 mL of complete medium. Twenty-four hours prior to transfection experiments, the medium was replaced with 1 mL of fresh culture medium with or without FBS. Liposomes 5/DNA complexes, equivalent to 1 μg of plasmid DNA at desired N/P ratios, were added to each well and incubated with cells for 4 h. The medium was then replaced with 0.5 mL of fresh complete medium, and incubated for further 24 h at 37 °C. For luciferase assays, cells were transfected by lipoplexes containing pGL3 plasmid DNA. The luciferase assay was performed using the Picagene luciferase assay kit (Tokyo Ink, Tokyo, Japan). The transfected cells were washed three times with phosphate-buffered saline (PBS) and lysed in a cell lysis buffer. The lysate was centrifuged at 4000 r/min at 4 °C for 2 min, and the supernatant was subjected to the luciferase assay. The relative light unit (RLU) of chemiluminescence was measured using a luminometer (Turner Designs, 20/20, Promega, Madison, WI, USA), and the luminescent RLU values were normalized to the protein content as determined by a BCA (bicinchoninic acid) assay. Transfection with lipofectamine 2000/DNA complexes and PEI-25K (N/P = 10, 1 μg DNA, diluted with Opti-MEM medium) were performed as positive controls. All transfection experiments were performed in triplicate.^11–13

Confocal laser scanning microscopy analysis

HeLa cells were seeded onto a 35-mm confocal dish (Φ = 15 mm) at a density of 2.5 × 10⁵ cells per well. After 24 h, the medium was replaced with fresh complete culture medium. Next, complexes of liposomes and Cy5-labeled pDNA (1 μg DNA per well) at a known concentration were added into each well. After incubation for 2 h, the cells were washed three times with PBS buffer, and the cell nuclei were stained with DAPI for 15 min. The cells were subsequently rinsed three times with PBS buffer. The confocal laser scanning microscopy (CLSM) imaging was performed using a Zeiss, LSM 780 (Zeiss, Japan) with a 40× objective lens at excitation wavelengths of 488 nm for liposome 5c (green), 650 nm for Cy5 (red), and 405 nm for DAPI (blue), respectively.

Statistical analysis

All the results are presented as the mean value ± SD. Paired or independent t-tests were performed to compare the differences between two groups. To meet the requirements (normal distribution and homogeneous variance) of the t-test, the data of luciferase expression were transformed by applying the base-10 logarithm. In addition, to determine whether the N/P was a factor affecting the cytotoxicity and luciferase expression, one-way analysis of variance (ANOVA) was performed. All statistical analyses were based on R 4.0.2; p < 0.05 was considered statistically significant.