Sunlight-Mediated Thiol–Ene/Yne Click Reaction: Synthesis and DNA Transfection Efficiency of New Cationic Lipids

Abstract

The design of green synthetic reaction conditions is very challenging, especially for biomaterials, but worthwhile if the compounds can be easily synthesized in the aqueous medium. Herein, we report the development of sunlight-mediated thiol–ene/yne click reaction in the presence of a catalytic amount of tert-butyl hydroperoxide (TBHP) in an aqueous medium. The optimized reaction conditions were successfully applied to synthesize a series of small molecules and lipids in a single step in the aqueous medium. The synthetic cationic lipid/co-lipid formed positively charged stable nanosized liposomes that effectually bind with the genetic materials. The in vitro DNA transfection and cellular uptake assays showed that the synthesized cationic lipids have comparable efficiency to commercially available Lipofectamine 2000. This mild synthetic strategy can also be used for smart design of novel or improvement of prevailing lipid-based nonviral gene delivery systems. Such chemical transformations in the aqueous medium are more environment-friendly than other reported thiol–ene/yne click reactions performed in an organic solvent medium.

Introduction

Over the past 2 decades, numerous viral or nonviral gene delivery systems have been developed to fight against various life-threatening diseases, including cancer, cardiovascular alignment, and others. Clinical studies have shown that the modification of cells at the genetic level significantly improves their therapeutic potentials.^1,2 The recently developed CRISPR/cas9-mediated genome engineering technology has extended the application of gene therapy to investigate the desirable and undesirable cellular processes.³ Nucleic acid delivery also provides a new perspective for the treatment of tandem healing, antitumor T cell immunity, cancer vaccines, and others.^1,3−6 However, a vehicle is required to encapsulate and transport the negatively charged genetic materials across the hydrophobic lipid bilayer for its successful delivery to the cells. Viral vectors-based delivery systems have been widely used for the successful delivery of genetic materials both under in vitro and in vivo conditions. Surprisingly, the random integration of the viral gene delivery system into the host genome interposes the target gene expression, which diminishes their therapeutic potentials. In this regard, nonviral gene delivery systems have been developed as an alternative approach.^1,3−9

Among the nonviral gene delivery systems, the natural/synthetic lipids are one of the most promising vehicles to optimize the delivery of exogenous genetic materials.^1,2,9,10 The lipids provide safeguard to genetic materials and obtain improved bioavailability with reduced immunogenicity and cytotoxicity. In particular, the cationic lipid-based vehicles show tremendous applications because of their efficient lipoplexes formation and membrane fusion capability.^1,2,10,11 Since the discovery of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (a cationic lipid with ammonium headgroup and with ether-linked hydrophobic tails), several lipids have been developed to augment the delivery of genetic materials.^{1,2,5,7,9,12−15} Few cationic lipid-based gene delivery vehicles are also commercially available (such as Lipofectamine 2000 and TransFast).¹⁶

A detailed structural investigation revealed that the general structure of the cationic lipids contains three crucial moieties, including a hydrophilic headgroup, a linker, and hydrophobic tails. The presence of ammonium moiety (or moieties) provides the required cationic charge to the headgroup, which is a prerequisite for enhanced electrostatic interactions with the phosphate backbone of the nucleic acids. The hydrophobic tails in association with the polar headgroup self-assembled in the aqueous medium to form liposomes capable of forming stable lipoplexes.^8,14,16−19 The linker region between the hydrophobic tails and hydrophilic headgroup provides additional means for their interactions with water and nucleic acids.^20,21 Unfortunately, the gene delivery efficacy of most of the reported cationic lipids is generally not adequate for clinical and other in vivo applications and also has shortcomings, including harsh reaction conditions with multistep synthetic routes, structural complexity, low stability, low transfection efficiency, and biocompatibility. Recent studies also demonstrated that the higher numbers of cationic ammonium moieties in the headgroup region also enhance their cytotoxic effect.^{1−3,7−11,14,16,17,21−24}

Currently, scientists are more focused on to make chemical bonds by utilizing various sources of renewable energies. The green chemical reactions, which take the benefits of plentiful, nonpolluting, and inexpensive solvent and renewable energy sources, such as water and sunlight, respectively, have been widely investigated for applications in biological science, organic chemistry, material sciences, and other related research areas.^6,25−29 The click reaction is considered as one of the successful green chemical reactions. The thiol–ene/yne click reaction has emerged as a valuable tool to synthesize macromolecules like gels, lipids, polymers, materials, and others. The synthesis of thioethers is also advantageous due to the high abundance of sulfur-containing natural products and bioactive molecules. The thiol–ene/yne click chemistry is an attractive and atom-economical approach for the construction of thioethers and is useful for efficient postpolymerization modification. The thiol–ene/yne click reaction generally proceeds either via thiol-Michael addition or via thermal/photochemical radical pathways. In the radical pathways, a photoinitiator or thermal initiator is essential to generate a radical and consequently produce the thiyl radicals from thiol compounds via hydrogen abstraction. The thiyl radical has the aptitude to couple with an alkene/alkyne.^{4,6,23,29−31}

Although the thiyl radical-mediated hydrothiolation process is very proficient and tolerant of various functional groups, it requires a photoinitiator or thermal initiators.⁴ Besides, most of the reported thiol–ene/yne click chemistry approaches suffer from harsh reaction conditions with the possible formation of disulfides as byproducts, which restricts its applications to only simple systems.^4,31,32 The thermal initiation reactions require a comparatively high temperature and longer time to accomplish the full conversion of the alkene/alkynes. The use of metal catalysts like Ru(II) complexes for the thiol–ene/yne click reaction offers excellent potential with higher stereo- and regioselectivity.³³ The UV/sunlight-mediated thiol–ene/yne click reaction in the presence of dyes like eosin-Y has also attracted widespread current research interest due to the green and sustainable process of syntheses.^25,34,35 Recent developments of photoredox catalysts have offered an attractive approach to generate thiyl radical in complex systems. However, most of these photoinduced reactions require expensive photosensitive catalysts to utilize the visible light in sensitizing the organic molecules.^{4,6,25−28,33,34,36,37} The use of expensive photoinitiators limits the applicability of these photochemical reactions in large-scale production of the synthetic materials.^30,37 Recently, it has been demonstrated that the thiols could be easily converted to thiyl radicals under sunlight at ambient temperature and this thiyl radical can undergo “click” reactions with alkene or alkyne.^25,29,31 Most importantly, the reported thiol–ene/yne click reactions are limited to organic solvents [like N,N-dimethylformamide (DMF), CH₂Cl₂, dioxane, and tetrahydrofuran (THF)] because of the poor stability of the thiyl radicals in the aqueous medium.^4,31−33,38 Because of the environmental concerns, the chemical reactions in the aqueous phase are much more superior to organic solvents. Synthesis of thioethers via thiol–ene/yne click reaction in aqueous medium continuously provides new challenges as well as prospects for chemists; also, there has been an increasing demand for green synthetic routes for the synthesis of organosulfur compounds in the development of drugs and materials application. The synthesis of materials, including polymers in the aqueous medium, is also useful in manufacturing cosmetics, pharmaceuticals, water purifications, oil separation, and others. We hypothesize that if thiyl radicals can be generated in situ in the aqueous medium, then the applications of thiol–ene/yne click chemistry will be manifold.

Herein, sunlight-mediated thiol–ene/yne click reaction in the presence of a catalytic amount of tert-butyl hydroperoxide (TBHP) in the aqueous medium was demonstrated. The optimized reaction conditions have been proven to be efficient and powerful for the synthesis of small molecules of chemical and pharmaceutical interest. This synthetic methodology was also applied to prepare a series of modular lipid molecules with neutral, zwitterionic, and cationic headgroups. The synthesized lipids form stable liposomes in the aqueous medium and possess several favorable physicochemical properties of lipids. Notably, these synthetic cationic lipids showed stronger DNA binding affinity and efficient DNA transfection ability with low cytotoxicity in different cell lines. Overall, this sunlight-mediated thiol–ene/yne click reaction is highly beneficial to synthesize small and complex molecules in the aqueous medium.

Results and Discussion

Optimization of the Reaction Conditions for the Thiol–Yne Click Reactions under Aqueous Environment

We started with mild reaction conditions to perform the thiol–ene/yne click reaction in an aqueous medium. The thiyl radicals are known to undergo click reactions with alkenes or alkynes in the absence or presence of radical initiators under an organic solvent.⁴ However, the formation of thiyl radicals in the aqueous medium is challenging. Hence, our first target was to generate thiyl radicals in the aqueous medium. We started our investigation with the model reaction of propargyl alcohol and dodecanthiol under sunlight in various reaction conditions, and the results are summarized in Table 1. The tert-butyl hydroperoxide (TBHP) is a water-soluble, inexpensive radical initiator and known to form t-BuO^• or t-BuOO^• radicals in the aqueous medium.³⁹ The oxidation of thiol by t-BuO^• or t-BuOO^• radicals could generate the thiyl radicals that can react with alkene/alkyne to produce the thioethers. To our delight, a catalytic amount of TBHP (0.05 mol %) as the radical initiator and H₂O as the solvent (Table 1, entry 17) led to the formation of the hydrothiolated product with the highest yields. Interestingly, redox additives, including Fe²⁺/Fe³⁺ and Cu⁺/Cu²⁺, adversely affected the model reaction with lower yields. The use of H₂O₂ and di-tert-butyl peroxide (DTBP) failed to produce the targeted product. Solvent screenings revealed that H₂O or H₂O/DMF (1:0.01) was producing very similar results to those in the presence of organic solvents (Table 1, entries 12–14 and 17). Therefore, to develop mild reaction conditions, a H₂O or H₂O/DMF solvent mixture was used. The reaction was also successfully performed in the presence of UV light (λ_ex = 365 nm), suggesting that in the absence of sunlight, the reaction can also be performed. Additionally, the reaction under dark conditions produces the targeted compound with a very low yield, which indicates that the light is necessary for the reaction. Moreover, a control reaction in the presence of (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) drastically reduced the reaction yield (Table 1, entry 20), which confirms that the reactions proceed through the radical pathway under the aqueous environment. The thiols and alkenes/alkynes are generally unreactive toward each other. However, various initiators can initiate their reactions. We hypothesized that the coupling of thiols with alkyne proceeds via a radical reaction pathway, and the TBHP acts as a thiyl radical initiator under aqueous medium. The exposure of sunlight initiates the cleavage of TBHP into tert-butoxyl and hydroxyl radicals.³⁹ Subsequently, the tert-butoxyl radical converts the thiols into thiyl radicals, and these thiyl radicals undergo coupling reaction with alkynes to produce the corresponding hydrothiolated product. The formation of tert-butoxyl and hydroxyl radicals under the optimized reaction conditions was confirmed by the high-resolution mass spectrometry (HRMS) analysis of the reaction mixture (Figure S1). Based on these experimental outcomes and control experiments, a proposed mechanism for this TBHP-initiated thiol–ene/yne click reaction is demonstrated in Figure 1.

Table 1. Optimization of the Reaction Conditions for the Synthesis of Thioethers via Thiol–Yne Click Reaction in the Aqueous Medium^c.

entry	solventa	initiator (mol %)	additive (mol %)	external stimulus	time (min)	yields (%)b
1	H2O	TBHP (1.0)	FeCl3 (0.5)	sunlight	30	77
2	H2O	TBHP (1.0)	FeSO4 (0.5)	sunlight	30	71
3	H2O	TBHP (1.0)	Fe(NO3)3 (0.5)	sunlight	30	72
4	H2O	TBHP (1.0)	CuCl (0.5)	sunlight	30	78
5	H2O		FeCl3 (0.5)	sunlight	30	ND
6	H2O		FeSO4 (0.5)	sunlight	30	ND
7	H2O		Fe(NO3)3 (0.5)	sunlight	30	ND
8	H2O		CuCl (0.5)	sunlight	30	ND
9	H2O	TBHP (1.0)		sunlight	30	85
10	H2O	H2O2		sunlight	30	10
11	H2O	DTBP		sunlight	30	80
12	DMF	TBHP (1.0)		sunlight	30	91
13	THF	TBHP (1.0)		sunlight	30	92
14	dioxane	TBHP (1.0)		sunlight	30	89
15	H2O	TBHP (0.5)		sunlight	30	83
16	H2O	TBHP (0.1)		sunlight	30	83
17	H2O	TBHP (0.05)		sunlight	30	91
18	H2O	TBHP (0.05)		UV light	30	90
19	H2O	TBHP (0.05)		dark condition	30	<10
20	H2O	TBHP (0.05)	TEMPO (0.5)	sunlight	30	ND

^aThe reaction was performed using 0.46 mmol 1a (1 equiv) and 1.0 mmol 2a (2.2 equiv) under sunlight.

^bYields of purified compounds.

^cND = not detected, DMF = N,N-dimethylformamide, THF = tetrahydrofuran.

Figure 1.

Proposed mode of reaction under optimized reaction conditions.

Synthesis of Small Molecules and Cationic Lipids

With the optimized reaction conditions in hand, we explored differently substituted alkenes and alkynes with various thiols for the application of this thiol–ene/yne click reaction under aqueous medium (Table 2). The reaction of aliphatic alkynes with thiols showed bis-hydrothiolation reactions with an excellent yield of 89–92% (compounds 3a–c). However, the anti-Markovnikov selective thiol–yne click reaction showed a monohydrothiolated product from aromatic alkynes (compounds 3d–f). The aliphatic and aromatic alkenes also showed excellent hydrothiolation reactions under the optimized reaction conditions (compounds 5a–g).

Table 2. Substrate Scopes for the Synthesis of Thioethers via Thiol–Yne/Ene Click Reactions^a.

^aThe reactions were performed using 0.046 mmol 2 or 4 and 1 mmol 1 in the presence of 0.05 mmol TBHP; the reactions for compounds 3a–c were performed in H₂O (2 mL); and the reactions for compounds 3d–f and 5a–g were performed in H₂O/DMF (2 mL) solvent mixture (1:0.01, v/v).

We also synthesized a series of lipids using the thiol–ene/yne click reaction under the optimized reaction conditions. We synthesized three different types of lipids with cationic, zwitterionic, and neutral headgroups.^31,40,41 The headgroups of lipid molecules were synthesized from a common starting molecule 2-((prop-2-yn-1-yloxy)methyl)oxirane (Scheme S1). The reaction of 2-((prop-2-yn-1-yloxy)methyl)oxirane (6) with the mono-/di-alkyl amine, followed by methylation of amine, resulted in the headgroup of the lipid molecules. The reaction of dimethylamine with 2-((prop-2-yn-1-yloxy)methyl)oxirane (6) resulted in alkynyl derivative 1-(dimethylamino)-3-(prop-2-yn-1-yloxy)propan-2-ol (7), which upon reaction with iodomethane and 2-bromoacetic acid yielded alkynyl derivatives 8 and 9, respectively. The reaction of compound 6 with 2,2′-azanediyldiethanol, followed by methylation, resulted in alkynyl derivative 11. The reaction of compound 6 with tert-butyl (2-aminoethyl)carbamate, followed by methylation and removal of the Boc group, produced the alkynyl derivative 14 (Scheme S1). After that, the reactions between these alkynyl headgroups with long-chain alkyl thiols were performed by using our optimized reaction conditions. The reactions of alkyl thiols of different chain lengths (C12 and C16) with alkynyl derivatives produced the lipids 15–19 (Figure 2 and Table 2). We also synthesized the control compound 20a (without the β-hydroxy group in the linker region) under similar reaction conditions (Table 2). The alkyl thioethers of the synthesized lipids are analogous to the two acyl chains present in the natural phospholipids. One of the significant challenges in the synthesis of most of the cationic lipids using the conventional chemistries is the typical obligation of protection and deprotection for the installation of acyl or ether linkage containing headgroups. Interestingly, the thiol–yne reaction produced the thioether-based lipids in the absence of protecting groups, suggesting that the presence of free acid, amine, and hydroxyl groups did not appear to obstruct the formation of lipids. The higher chemoselectivity of the thiol–yne reaction could be one of the reasons for the efficient synthesis of these lipids.⁴¹ It is important to mention that for the synthesis of compounds 15–20, the H₂O/DMF solvent mixture (1:0.01) was used to increase the solubility of dodecanethiol or hexadecanethiol. However, the reaction may also proceed at the interface of the aqueous medium. Overall, we successfully synthesized a series of lipids using an inexpensive and environment-friendly approach.

Figure 2.

Rapid synthesis of dithioether-based cationic lipids through sunlight-mediated thiol–yne clicks reactions in the aqueous medium. The reactions were performed using 0.46 mmol 7–11 (1 equiv) and 1.0 mmol RSH (2.2 equiv) in the presence of 0.05 mmol TBHP in 2 mL of H₂O/DMF (1:0.01, v/v) under sunlight.

Physicochemical Properties of the Cationic lipids

We observed that hydration of the thin films of the lipids forms stable aggregates in aqueous solution. Transmission electron microscopy (TEM) analyses were performed to investigate the aggregation pattern of the lipids in an aqueous medium.^16,42−45 The TEM images revealed the formation of small and medium-sized liposomes (200–450 nm) by these lipids (Figure 3). We characterized the physicochemical properties such as particle size, surface potential, phase-transition temperature, and DNA-binding properties of the lipids. The dynamic light scattering (DLS) measurements also showed the formation of vesicles in an aqueous medium with a very similar hydrodynamic diameter and narrow size distribution (Figure 4A). The hydrodynamic diameters of the liposomes obtained by the DLS measurements of the lipids were found to be within 200–450 nm.^42,43 The lipids with shorter chain length formed liposomes with smaller sizes. We also examined the effect of pH on the hydrodynamic diameter and surface charge of the lipids. The DLS measurements showed that the pH of the environment has little impact on the hydrodynamic diameter of the lipids 16a and 19a (Figure 4B).^16,42,43 However, the pH of the environment plays a vital role in the surface charge of the lipids, which could be due to the presence of ammonium ion in the lipid headgroup (Figure 4C). The intramolecular ionic interaction could be one of the reasons for the deprotonation of secondary alcohol around pH 8.0. The isoelectric points (IEPs) of the lipids 16a and 19a were 8.19 and 8.09, respectively. The IEP values also demonstrate that these lipids are generally cationic under physiological conditions.

Figure 3.

TEM images of the vesicles generated from 100% lipids of 15–19a (A–E) and 15–19b (F–J). For lipids 15–19a and 15–19b, the chain lengths were C12 and C16.

Figure 4.

Mean particle size of the lipid vesicles at pH 7.4 (A). Mean particle size (B) and surface potential (C) of the lipids 16a and 19a at different pH solutions. All measurements were performed at room temperature. Temperature-dependent steady-state fluorescence anisotropy measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) under the vesicle environment for 16a and 19a lipids within the range of 10–80 °C (D). The inset shows the T_m values of the lipids.

Fluidity and the transition from the gel phase to the liquid disordered phase are essential parameters of the lipids. Hence, the phase-transition temperature (T_m) of the cationic lipids can be considered as a fundamental physical property to investigate the thermal stability and gene delivery properties of the liposomes.^42,43 We performed temperature-dependent steady-state anisotropy measurements using the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence probe to determine the T_m values of the lipids (Figure 4D). The observed T_m values of lipids 16a and 19a were 43 and 37 °C, respectively. This elevation of T_m values could be due to the more ordered packing in the plane of the lipid bilayer.^42,43 This moderate thermal stability also suggests that these lipids could have multiple biological applicability. We also assume that in the presence of fusogenic co-lipid such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PE), the liposomes would also be stable.

Interaction of the Lipids with the Plasmid DNA

The liposomes of these synthetic lipids were prepared in combination with co-lipid PE (1:1 mole ratio). The inverted hexagonal structure of PE lipid is known to facilitate the endosomal membrane fusion and facile release of DNA, leading to higher transfection efficiencies of the lipoplexes.^8,16,31 The DNA-binding capability is the prerequisite for gene delivery vectors, and the optimum balance between the DNA binding and release is imperative to enhance transfection efficiency. The DNA binding ability of these liposomes was investigated by electrophoretic gel retardation assay. The green fluorescence protein (GFP)-tagged plasmid DNA of the MMP7 enzyme (pMMP7-GFP) was used for all gene delivery-related studies. Efficient gene delivery vehicles should bind plasmid DNA at low N/P ratios. The outcome of the gel retardation studies showed that both hydrophobic chain length and overall charge of the headgroup have a variable effect on their DNA-binding ability (Figure 5). The mobility of the plasmid DNA was strongly inhibited in the presence of the lipids with monocationic headgroups (16 and 19) at the N/P ratios of 1–2. The lipids with zwitterionic headgroup (18) failed to retard the plasmid DNA up to the N/P ratio of 8, indicating their weaker DNA binding ability. The cationic and polyamine-containing lipids are known to have stronger DNA-binding ability.^9,16,24 Surprisingly, the lipid 17 with ammonium and trialkylammonium headgroups showed lower plasmid DNA-binding ability (N/P ratio ≥2), suggesting that the presence of the β-hydroxy group in the linker region plays a vital role in their DNA-binding abilities. The hydroxy group may interact with the terminal ammonium group of lipid 12, leading to its inaccessibility for DNA binding.¹⁵ We also synthesized lipid (20a) without the β-hydroxy group-containing linker region. The gel retardation studies showed a much weaker DNA-binding ability (even N/P > 4) of lipid 20a, suggesting the importance of the β-hydroxy group in DNA binding (Figures S2 and S3). Overall, the cationic lipids showed stronger DNA-binding abilities with C12 in comparison to that of C16 chain length, suggesting that the length of the hydrophobic alkyl group is also a key parameter in stable lipoplex formation. Hence, the plasmid DNA-binding affinity of the synthesized lipids follows an order of 19a ≈ 16a ≈ 16b > 19b ≈ 17a > 15a > 15b ≈ 17b ≈ 18a ≈ 18b, suggesting that the DNA-binding affinity of the lipids can be tuned by choosing certain types of headgroup and hydrophobic tails. For cellular applications, the lipoplexes should be stable enough to evade possible detachment under the physiological conditions before they reach the targeting tissues. In this regard, the DNA-binding ability of the potent cationic lipids 16 and 19 was investigated in the presence of serum.¹⁶ The gel retardation assay showed that these cationic lipids also effectively inhibited the mobility of plasmid DNA at N/P ratio of 2–4 in the presence of both 10 and 50% serum, suggesting that the presence of serum has a minimal effect on their DNA-binding abilities (Figure S4). The time-dependent studies showed no release or enzymolysis of DNA even after 2 h, suggesting the stability of the lipoplexes in the presence of serum (Figure S4). It is important to mention that the appearance of one or two bands with different brightnesses in the gel images could be due to the change in the structure of the DNA after the formation of the lipoplexes.

Figure 5.

Electrophoretic gel retardation assay of pMMP7-GFP in the absence and presence of co-liposomes at different N/P ratios (0–8). The lipids of C12 (A–E) and C16 (F−J) chain lengths and PE were used to measure their DNA-binding abilities by gel electrophoresis assay. The molar ratio of synthetic lipid and PE was 1:1 (mole ratio). All of the gel electrophoresis measurements were performed in triplicates.

Relation between the Particle Size and Surface Charge of the Lipoplexes

The gene transfection efficiency greatly depends on the size and surface charge of the lipoplexes, which depends on the structural properties of headgroup and hydrophobic tails. In this regard, the hydrodynamic diameter and surface charge of the lipoplexes were investigated. The outcome of the DLS measurements showed that the average d_H’s of the liposomes and lipoplexes were within 200–500 nm (Figure 6A,C). The variable sizes of the liposomes suggest that the hydrophilic properties of the headgroup played an essential role in the liposome formation. The lipids with shorter chain lengths could produce lipoplexes with a small size. The liposomes of 16, 17, and 19 have a positive surface charge, which facilitates their electrostatic interaction with the negatively charged DNA. The lipids 15 and 18 formed negatively charged liposomes under physiological conditions. Meanwhile, different surface charges were observed for the lipoplexes at various N/P ratios (Figure 6B,D). The lipoplexes of 16, 17, and 19 demonstrated a rapid increase in the surface charge when the N/P ratios were varied from 0.5 to 8. The lipoplexes of 16 and 19 have a cationic surface potential, and average d_H values were less than 400 nm at an N/P ratio of 2, at which full retardation of DNA was observed in the gel electrophoresis assay. The hydrophobic chain length had no substantial effect on both size and surface charge of the lipoplexes. The lipoplex of 15 showed a slower increases in surface charge, but the lipoplex of 18 showed a decrease in surface charge within the same range of N/P ratios, suggesting that the surface charge could be tuned by selection of a particular lipid structure. The outcome of the size and surface charge measurements suggests that the lipoplexes might possess a different cellular gene transfection efficiency. The TEM analysis was also performed to investigate the morphologies of the lipoplexes at an N/P ratio of 2 (Figures S5 and S6). The TEM images showed that both the lipids could adequately encapsulate the DNA. However, the size of the lipoplexes obtained from the TEM images of 16 and 19 (50–200 nm) are much smaller than that measured by DLS analysis (200–400 nm), which could be due to the shrinking of lipoplexes in the drying process during the TEM sample preparation, while the DLS measures the hydrodynamic sizes in the aqueous solution. The lipoplexes formed from lipid 16a showed a larger size than other lipids.¹⁶

Figure 6.

Mean particle size (A, C) and surface potential (B, D) of the lipoplexes at different N/P ratios. The lipids of C12 (A, B) and C16 (C, D) chain lengths were used to measure the particle size and surface potential of the corresponding lipoplexes. DLS measurements were performed at room temperature. The molar ratio of synthetic lipid and PE was 1:1.

Gene Transfection Efficiency of the Lipoplexes

The cytotoxicity of the lipids was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using human embryonic kidney 293 (HEK-293T) and baby hamster kidney fibroblasts (BHK-21) cell lines.³¹ The lipids 16a, 17a, and 19a were selected for investigating their applicability because of their higher DNA-binding abilities. The MTT results showed that the IC₅₀ values of the lipids were within 106–165 and 80–144 μM for HEK-293T and BHK-21 cell lines, respectively (Figure S5 and Table S1). Overall, the IC₅₀ values indicate the low toxicity of these lipids, which is useful for their successful application as a nonviral gene delivery system.

The outcome of the gel retardation assay and the formation of stable lipoplex suggest that the synthesized cationic lipids have the potential to deliver the genetic material to cells. Hence, in vitro gene transfection efficiencies of the lipids were investigated by fluorescence microscopy assay in HEK-293T cells using pMMP7-GFP plasmid DNA encoding the GFP-tagged MMP7 enzyme. It is well documented that fusogenic lipid-like PE is usually required for effective transfection by the cationic lipids.^16,23,31 The synthetic lipids 16a, 17a, and 19a showed strong DNA-binding abilities (with N/P ratio >2) among all of the tested lipids. In this regard, the cellular assays were performed using only these potent lipids. The liposomes were prepared in combination with co-lipid PE (1:1 mole ratio). The liposomes with helper lipid-like PE show higher transfection and lower cytotoxicity than those without PE.^16,46 The lipoplexes were prepared at an N/P ratio of 2–8 using pMMP7-GFP DNA as the reporter gene, and Lipofectamine 2000 was used as the positive control (Figures 7 and S7). The cellular expression of GFP protein was monitored to measure the transfection efficiency of the lipids. The outcome of the gene transfection experiment revealed that lipids 16a, 17a, and 19a (at an N/P ratio of 1–4) had a comparable transfection efficiency to the commercially available Lipofectamine 2000 (Figures S8 and S9). The lipids with C16 chain lengths showed comparatively weak DNA-binding efficiencies. Hence, the transfection efficacies of those lipids were not investigated. The presence of unsaturated hydrophobic tails causes weak intermolecular hydrophobic interactions, leading to the formation of less stable liposomes, which generally does not have any significant effect on the DNA binding and delivery efficacies; hence, those classes of lipids were also not investigated.^16,42,43 The final concentrations of the lipids used for the delivery of pMMP7-GFP DNA were within 15–120 nM (for N/P ratios 1–8), which are much lower than their IC₅₀ values (106–165 μM; obtained from the MTT assay). Hence, the toxicity of these lipids could have a negligible impact on their gene transfection efficiencies.

Figure 7.

Fluorescence microscopy images of the MMP7-GFP expression in HEK-293T at various N/P ratios. Scale bar: 100 μm. Lipofectamine 2000 was used as control. Images were recorded using an inverted fluorescence microscope.

We also evaluated the cellular uptake of pMMP7-GFP in HEK-293T cells at different N/P ratios. The internalization of the lipoplexes significantly influences the transfection efficiency of the lipids. We performed the flow cytometry assay to investigate the cellular uptake of the DNA.¹⁶ After transfection, the percentage of cells positive for GFP-labeled pMMP7 was calculated and is shown in Figures 8, S10, and S11. For fluorescence-activated cell sorting (FACS) analyses, experiments were performed using the FL1 channel for green fluorescence, and the data were analyzed using FCS Express 6 Flow Cytometry software. The autofluorescence was detected using nontransfected cells as control indicated as a peak in the histogram plot using the FL1 channel. The M1 region is indicated in plots capturing green fluorescence intensity in transfected cells, which is more than autofluorescence intensity. The percentages of GFP positive cells were calculated to measure the cellular uptake properties of the lipids. The outcome of the flow cytometry analysis showed that there was only a little difference between 16a, 19a (N/P = 2 and 4), and Lipofectamine 2000, indicating that the lipoplexes of these synthetic lipids have comparable intracellular delivery ability. The cellular uptake efficiencies of lipids 16a and 19a were higher than that of 17a, which is in good agreement with their DNA-binding and transfection efficiencies. However, the transfection efficiencies of all of the tested lipids decrease with N/P ratio >4. A similar consequence was observed for their DNA transfection efficiencies, suggesting that very high positive surface potentials limit their transfection abilities in healthy HEK-293T cells. The outer membrane of the normal healthy cells is generally zwitterionic because of the high abundance of zwitterionic lipids like phosphatidylcholine and others.⁴⁷ We are currently performing comprehensive transfection mechanism studies of such types of lipids in different cell lines.

Figure 8.

Cellular uptake of lipoplexes (pMMP7-GFP) at different N/P ratios in HEK-293T cells after 24 h of transfection. The percentages of cellular uptake efficiencies were quantified by flow cytometry analysis.

Conclusions

The sunlight-mediated synthetic reaction conditions in the aqueous medium have been developed to synthesize a series of small molecules and lipids. The thiol–yne/ene click reaction was performed in the aqueous medium in the presence of a catalytic amount of tert-butyl hydroperoxide (TBHP) with a very high hydrothiolation efficiency. The biophysical properties of the lipids, such as particle size, surface potential, phase-transition temperature, and DNA-binding ability, were thoroughly investigated. Most importantly, all of these phosphorous-free lipids form stable vesicles, but only the cationic lipids form stable lipoplexes. The structure-based DNA-binding studies of the lipids revealed that the presence of the β-hydroxy group in the linker region plays a vital role in forming stable lipoplexes. Additional studies also revealed that the hydrophobic chain length significantly affects the DNA-binding abilities of the lipids, and C12 was found to be more suitable than C16 chain length. The potent cationic lipids displayed much lower cytotoxicity. Interestingly, these cationic lipids also showed comparable gene transfection and cellular uptake properties with the commercially available Lipofectamine 2000, signifying their prospective to be favorable nonviral gene vectors. Therefore, the design and synthesis of such sunlight-driven cationic lipids may be a promising approach for lipid-based gene delivery systems. However, further optimization of the lipid structure to improve the gene delivery activities is required to establish their in vivo applications. Overall, such mild reaction conditions can be applied for the smart design of new or improvement of existing lipid-based gene delivery systems. This synthetic strategy can also be used to synthesize other small or macromolecules for pharmaceutical materials and others in the aqueous medium.

Experimental Section

General Information

All reagents were purchased from Sigma-Aldrich, Merck, Himedia, and used directly without further purification. Column chromatography was performed using 60–120 mesh silica gels. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ (0.25 mm). ¹H NMR and ¹³C NMR spectra were recorded at 400 and 100 MHz, respectively, with a Varian AS400 spectrometer and 600 and 151 MHz with Brucker spectrometers, using tetramethylsilane (TMS) as an internal standard with CDCl₃, dimethyl sulfoxide (DMSO)-d₆. The coupling constants (J values) and chemical shifts (δ_ppm) were reported in hertz (Hz) and parts per million (ppm), respectively, downfield from tetramethylsilane using residual chloroform (d = 7.24 for ¹H NMR, d = 77.23 for ¹³C NMR) as an internal standard. Multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broadened). High-resolution mass spectroscopy (HRMS) images were recorded on an Agilent Q-TOF mass spectrometer with Z-spray source using built-in software for analysis of the recorded data. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (PE) was purchased from Avanti Polar Lipids (Alabaster, AL). Ultrapure water (Milli-Q system, Millipore, Billerica, MA) was used for the preparation of buffers.

General Procedure for the Thiol–Ene/Yne Click Reaction

All of the compounds were synthesized using the optimized reaction condition (Table 1, entry 17). To a solution of alkynyl/alkenyl derivative (0.46 mmol) in water (2 mL) or H₂O/DMF (10:0.5) solution mixture (2 mL) in a glass vial were added thiol derivative (1.0 mmol) and TBHP (0.023 mmol) solution and vortexed for 2 min. The reaction vial was then exposed to sunlight (weather condition: sunny, temperature, 30–36 °C) and kept for 30 min. After completion of the reaction, the mixture was diluted with cold water and ethyl acetate. The organic layer was extracted and washed with brine and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure. The reaction mixture was purified by column chromatography using EtOAc/hexane (2–40%) solvent gradient.

Synthesis of 2,3-Bis(dodecylthio)propan-1-ol (3a)

Following the general procedure (in water), using propargyl alcohol (40 mg, 0.71 mmol) and dodecanthiol (318 mg, 1.57 mmol) provided 300 mg (94% yield, time = 30 min) of 3a as a colorless liquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.81–3.78 (m, 1H), 3.67–3.64 (m, 1H), 2.91–2.87 (m, 1H), 2.82–2.79 (m, 1H), 2.71–2.67 (m, 1H), 2.57–2.51 (m, 4H), 1.59–1.54 (m, 4H), 1.39–1.32 (m, 4H), 1.30–1.24 (m, 33H), 0.86 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 63.3, 48.8, 35.0, 33.1, 32.0, 31.2, 30.1, 29.8, 29.7, 29.6, 29.5, 29.3, 29.0, 29.0, 22.8, 14.20; HRMS [electrospray ionization (ESI)] calcd for C₂₇H₅₆O₂S [M + K]⁺: 499.3614, found: 499.3622.

Synthesis of 2,3-Bis(dodecylthio)propan-1-amine (3b)

Following the general procedure (in water), using propargylamine (40 mg, 0.72 mmol) and dodecanthiol (324 mg, 1.59 mmol) provided 305 mg (92% yield, time = 30 min) of 3b as a yellowish semiliquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.03–3.00 (m, 1H), 2.82–2.81 (m, 3H), 2.66–2.62 (m, 1H), 2.55–2.48 (m, 4H), 1.58–1.53 (m, 4H), 1.36–1.32 (m, 4H), 1.28–1.23 (m, 32H), 0.85 (t, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 49.6, 44.3, 35.9, 33.2, 32.0, 31.1, 30.1, 29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.1, 29.0, 22.8, 14.2; HRMS (ESI) calcd for C₂₇H₅₇NS₂ [M + H]⁺: 460.4005, found: 460.4005.

Synthesis of 2,3-Bis(dodecylthio)butane-1,4-diol (3c)

Following the general procedure (in water), using but-2-yne-1,4-diol (40 mg, 0.46 mmol) and dodecanthiol (207.1 mg, 1.02 mmol) provided 201 mg (89% yield, time = 30 min) of 3c as a colorless liquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.88–3.74 (m, 2H), 3.06–2.84 (m, 4H), 2.68–2.60 (m, 2H), 2.58–2.51 (m, 2H), 1.68–1.47 (m, 4H), 1.37–1.31 (m, 4H), 1.29–1.25 (m, 36H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 62.3, 51.5, 32.7, 31.9, 30.0, 29.6, 29.6, 29.6, 29.5, 29.3, 29.2, 29.2, 28.8, 22.6, 14.1; HRMS (ESI) calcd for C₂₈H₅₈O₂S₂ [M + Na]⁺: 513.3770, found: 513.3770.

Synthesis of Dodecyl(1-phenylvinyl)sulfane (3d)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using ethynylbenzene (40 mg, 0.39 mmol) and dodecanthiol (174.56 mg, 0.86 mmol) provided 102 mg (85% yield, time = 30 min) of 3d as a colorless liquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 7.47–7.46 (d, 1H), 7.34–7.31 (m, 1H), 7.25–7.22 (m, 1H), 7.18–7.14 (m, 2H), 6.70 (d, J = 18 Hz, 1H), 6.43 (d, J = 18 Hz, 1H), 2.78–2.74 (m, 2H), 1.69–1.64 (m, 2H), 1.43–1.36 (m, 2H), 1.31–1.24 (m, 18H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 137.3, 128.7, 128.3, 127.9, 126.9, 126.7, 125.6, 125.5, 36.1, 32.8, 32.1, 30.4, 29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.0, 28.8, 22.9, 14.3; HRMS (ESI) calcd for C₂₀H₃₂S [M + H]⁺: 305.2297, found: 305.2305.

Synthesis of 2,3-Bis(dodecylthio)-3-phenylpropan-1-ol (3e)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using 3-phenylprop-2-yn-1-ol (40 mg, 0.30 mmol) and dodecanthiol (135 mg, 0.66 mmol) provided 91 mg (90% yield, time = 30 min) of 3e as a colorless liquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 7.63–7.59 (m, 2H), 7.36–7.32 (m, 2H), 7.26–7.22 (m, 1H), 6.82 (s, 1H), 4.34 (s, 4H), 2.77–2.66 (m, 2H), 1.64–1.50 (m, 2H), 1.33–1.29 (m, 2H), 1.25–1.22 (m, 17H), 0.88 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 136.2, 135.6, 129.8, 129.6, 129.4, 128.0, 127.3, 66.9, 31.9, 31.3, 29.8, 29.6, 29.5, 29.4, 29.3, 29.1, 28.7, 22.6, 14.1; HRMS (ESI) calcd for C₂₁H₃₄OS [M + H]⁺: 335.2403, found: 335.2416.

Synthesis of 2-((2-Hydroxyethyl)thio)-3-phenylprop-2-en-1-ol (3f)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using 3-phenylprop-2-yn-1-ol (40 mg, 0.30 mmol) and 2-mercaptoethanol (28.4 mg, 0.36 mmol) provided 58 mg (92% yield, time = 30 min) of 3f as a yellowish semiliquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.27–2.82 (m, 2H), 2.77–2.64 (m, 1H), 1.87–1.60 (m, 2H), 1.49–1.31 (m, 3H), 1.34–1.25 (m, 17H), 0.89 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 135.8, 134.3, 132.0, 129.3, 128.4, 128.2, 127.6, 127.4, 67.4, 61.2, 34.7; HRMS (ESI) calcd for C₁₁H₁₄O₂S [M + Na]⁺: 233.0607, found: 233.0607.

Synthesis of 3-(Dodecylthio)-2-methylpropanoic Acid (5a)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using methacrylic acid (40 mg, 0.46 mmol) and dodecanthiol (112.9 mg, 0.55 mmol) provided 119 mg (90% yield, time = 30 min) of 5a as a white solid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.27–2.82 (m, 2H), 2.77–2.64 (m, 1H), 1.87–1.60 (m, 2H), 1.49–1.31 (m, 3H), 1.34–1.25 (m, 17H), 0.89 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm; HRMS (ESI) calcd for C₁₆H₃₂O₂S [M + Na]⁺: 311.2015, found: 311.2016.

Synthesis of 2-(Dodecylthio)butane-1,4-diol (5b)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using but-2-ene-1,4-diol (40 mg, 0.45 mmol) and dodecanthiol (110 mg, 0.54 mmol) provided 114 mg (87% yield, time = 30 min) of 5b as a white solid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.09–4.03 (m, 1H), 3.87–3.77 (m, 1H), 3.08–3.05 (m, 1H), 3.01–2.73 (m, 1H), 2.32–2.19 (m,1H), 2.00–1.92 (m, 1H), 1.88–1.59 (m, 2H), 1.52–1.39 (m, 1H), 1.32–1.25 (m, 16H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 61.5, 60.0, 52.2, 31.9, 29.7, 29.6, 29.5, 29.3, 29.2, 29.0, 28.8, 28.5, 27.6, 22.9, 22.6, 21.2, 14.0; HRMS (ESI) calcd for C₁₆H₃₄O₂S [M + Na]⁺: 313.2172, found: 313.2172.

Synthesis of Dodecyl(phenethyl)sulfane (5c)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using styrene (40 mg, 0.38 mmol) and dodecanthiol (93 mg, 0.46 mmol) provided 99 mg (85% yield, time = 30 min) of 5c as a colorless liquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 7.31–7.29 (m, 2H), 7.23–7.20 (m, 3H), 2.90–2.87 (m, 2H), 2.78–2.76 (m, 2H), 2.54–2.52 (m, 2H), 1.59–1.57 (m, 2H), 1.39–1.34 (m, 2H), 1.31–1.26 (m, 17H), 0.88 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 140.9, 128.6, 126.5, 36.6, 33.8, 32.5, 32.1, 29.8, 29.8, 29.8, 29.7, 29.5, 29.4, 29.1, 22.9, 14.3; HRMS (ESI) calcd for C₂₀H₃₄S [M + H]⁺: 307.2454, found: 307.2457.

Synthesis of Dodecyl(4-methylphenethyl)sulfane (5d)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using 1-methyl-4-vinylbenzene (40 mg, 0.33 mmol) and dodecanthiol (82 mg, 0.40 mmol) provided 89 mg (85% yield, time = 30 min) of 5d as a colorless semiliquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 7.14–7.11 (m, 4H), 2.88–2.85 (m, 2H), 2.78–2.76 (m, 2H), 2.56–2.53 (m, 2H), 2.34 (s, 3H), 1.63–1.58 (m, 2H), 1.47–1.32 (m, 2H), 1.35–1.29 (m, 18H), 0.91 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 137.7, 135.7, 129.1, 128.3, 36, 33.8, 33.2, 31.9, 29.7, 29.7, 29.6, 29.6, 29.5, 29.3, 29.2, 28.9, 22.7, 21.0, 14.1; HRMS (ESI) calcd for C₂₁H₃₆S [M + H]⁺: 321.2610, found: 321.2612.

Synthesis of 2-((4-Methylphenethyl)thio)ethanol (5e)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using ethynylbenzene (40 mg, 0.39 mmol) and 2-mercaptoethanol (35.98 mg, 0.46 mmol) provided 61 mg (87% yield, time = 30 min) of 5e as a light yellowish semiliquid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 7.34–7.31 (m, 2H), 7.24–7.20 (m, 3H), 3.71 (t, 2H), 2.91–2.89 (m, 2H), 2.81–2.78 (m, 2H), 2.73 (t, 2H), 2.26 (s, 1H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 140.2, 128.5, 128.5, 126.6, 60.3, 36.3, 35.5, 33.2; HRMS (ESI) calcd for C₁₀H₁₄OS [M + K]⁺: 221.0569, found: 221.0591.

Synthesis of 3-((4-Methylphenethyl)thio)propanoic Acid (5f)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using 1-methyl-4-vinylbenzene (40 mg, 0.33 mmol) and 3-mercaptopropanoic acid (42 mg, 0.39 mmol) provided 66 mg (89% yield, time = 30 min) of 5f as a white solid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.27–2.82 (m, 2H), 2.77–2.64 (m, 1H), 1.87–1.60 (m, 2H), 1.49–1.39 (m, 3H), 1.34–1.25 (m, 17H), 0.89 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 177.9, 137.2, 135.9, 129.1, 128.3, 35.7, 35.6, 35.5, 33.7, 26.9, 21.0, 19.6; HRMS (ESI) calcd for C₁₂H₁₆O₂S [M + H]⁺: 225.0944, found: 225.0942.

Synthesis of (1S)-((1S,2S,4S)-5-(2-(Dodecylthio)ethyl)quinuclidin-2-yl)(6-methoxyquinolin-4-yl)methanol (5g)

Following the general procedure [in H₂O/DMF solvent mixture (1:0.01; v/v)], using quinine (40 mg, 0.12 mmol) and dodecanthiol (30 mg, 0.15 mmol) provided 56 mg (87% yield, time = 30 min) of 5g as a white solid; ¹H NMR (600 MHz, CDCl₃) δ_ppm 8.68–8.66 (m, 1H), 7.93–7.91 (m, 1H), 7.52–7.49 (m, 2H), 7.40–7.37 (m, 1H), 5.28–5.27 (d, 1H), 3.90 (s, 3H), 3.28–3.18 (m, 1H), 3.07–3.01 (m, 1H), 2.88–2.82 (m, 1H), 2.44–2.37 (m, 4H), 1.81 (s, 2H), 1.71–1.63 (m, 4H), 1.55–1.52 (m, 2H), 1.46–1.44 (m, 2H), 1.23–1.21 (m, 21H), 0.85–0.81 (m, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 157.2, 149.7, 147.9, 144.3, 131.5, 127.4, 121.4, 119.5, 102.9, 71.2, 60.8, 57.7, 56.2, 55.9, 52.0, 42.3, 34.8, 31.7, 31.5, 29.7, 29.5, 29.4, 29.3, 29.3, 29.1, 29.0, 28.9, 28.5, 28.4, 25.7, 25.5, 23.9, 23.1, 22.5, 14.3; HRMS (ESI) calcd for C₃₂H₅₀N₂O₂S [M + H]⁺: 527.3666, found: 527.3666.

Synthesis of 1-(Dimethylamino)-3-(prop-2-yloxy)propan-2-ol (7)

To a stirring solution of 2-((prop-2-yn-1-yloxy)methyl)oxirane (6) (8.92 mmol) in dry dichloromethane solvent dimethylamine (26.78 mmol) was added, and the resulting mixture was stirred for 12 h at room temperature under N₂ atmosphere. The progress of the reaction was monitored by TLC. After maximum consumption of compound 6, the solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel using a gradient solvent system of dichloromethane and methanol (10:0.6) to afford compound 7 as a yellow liquid. Characterization of compound 7: yellow liquid (yield 73%); ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.15 (d, J = 2.5 Hz, 2H), 3.92–3.86 (m, 1H), 3.84 (m, 1H), 3.54–3.51 (m, 1H), 3.45 (dd, J = 9.8, 5.7 Hz, 1H), 2.42 (m, 2H), 2.25 (m, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 79.5, 74.7, 72.3, 66.6, 61.9, 58.6, 45.5; HRMS (ESI) calcd for C₈H₁₅NO₂ [M + H]⁺: 158.1136, found: 158.1126.

Synthesis of 2-Hydroxy-N,N,N-trimethyl-3-(prop-2-yn-1-yloxy)propan-1-aminium (8)

To a stirring solution of 1-(dimethylamino)-3-(prop-2-yloxy)propan-2-ol (7, 0.827 mmol) in acetonitrile (10 mL) was added K₂CO₃ (0.827 mmol), and the reaction mixture was continued to stir for 30 min at room temperature. After that, iodomethane (0.82 mmol) was added, and the reaction mixture was heated under reflux condition for 12 h. After maximum consumption of compound 7, the reaction mixture was cooled down to room temperature and unused K₂CO₃ was removed by filtration. Removal of organic solvent under reduced pressure yielded an oily crude product. The addition of diethyl ether to this oily crude product yielded the target product as a colorless crystalline solid, which was used without further purification. Characterization of compound 8: colorless solid (yielded 87%); ¹H NMR (600 MHz, DMSO-d₆) δ_ppm 4.25 (m, 1H), 4.20–4.19 (m, 2H), 3.50 (t, J = 2.4 Hz, 1H), 3.48–3.46 (m, 1H), 3.39–3.33 (m, 3H), 3.15 (s, 9H); ¹³C NMR (151 MHz, DMSO-d₆) δ_ppm 80.5, 78.1, 72.3, 68.5, 64.4, 58.4, 53.0; HRMS (ESI) calcd for C₉H₁₈NO₂ [M]⁺: 172.1332, found: 172.1341.

Synthesis of N-(Carboxymethyl)-2-hydroxy-N,N-dimethyl-3-(prop-2-yn-1-yloxy)propan-1-aminium (9)

To a stirring solution of 1-(dimethylamino)-3-(prop-2-yloxy)propan-2-ol (7, 1.27 mmol) in dichloromethane (10 mL) was slowly added (dropwise) a solution of bromoacetic acid (3.82 mmol) in dichloromethane (2 mL) at room temperature under N₂ atmosphere. The resulting reaction mixture was stirred for 48 h; after that, a gummy part was observed below the reaction solution. Removal of organic solvent under reduced pressure yielded a gummy mass, which was washed with diethyl ether several times. The solvent (diethyl ether) wash yielded the target product, which was used without further purification. Characterization of compound 9: light yellow solid (yield 85%); ¹H NMR (400 MHz, CDCl₃) δ_ppm 4.22–4.17 (m, 1H), 4.16 (d, J = 2.3 Hz, 2H), 3.74–3.73 (m, 2H), 3.66–3.62 (m, 1H), 3.56–3.52 (m, 1H), 3.46–3.42 (m, 2H), 3.34–3.30 (m, 1H), 3.23–3.20 (m, 6H); ¹³C NMR (101 MHz, CDCl₃) δ_ppm 170.8, 85.2, 82.8, 77.1, 70.6, 69.1, 63.1, 56.5, 56.3; HRMS (ESI) calcd for C₁₀H₁₈NO₄ [M]⁺: 216.1230, found: 216.1231.

Synthesis of 2,2′-((2-Hydroxy-3-(prop-2-yn-1-yloxy)propyl)azanediyl)bis(ethan-1-ol) (10)

To a stirring solution of compound 6 (4.42 mmol) in dichloromethane (10 mL) was added a solution of diethanolamine (8.84 mmol) in dichloromethane (2 mL) at room temperature under N₂ atmosphere. The resulting reaction mixture was stirred for 8 h. After completion of the reaction, the unused solvent was removed under reduced pressure. The yellowish crude reaction mixture was purified by column chromatography on silica gel using a gradient solvent system of dichloromethane and methanol (10:1.2). Characterization of compound 10: light yellow viscous liquid (yield 82%); ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.82 (brs, 3H), 4.17–4.16 (m, 2H), 3.93–3.88 (m, 1H), 3.70–3.66 (m, 2H), 3.53–3.45 (m, 4H), 2.75–2.70 (m, 2H), 2.59–2.54 (m, 1H), 2.44–2.37 (m, 4H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 79.5, 74.8, 72.1, 67.5, 59.3, 58.6, 58.3, 57.3; HRMS (ESI) calcd for C₈H₁₅NO₂ [M + H] ⁺: 232.1543, found: 232.1539.

Synthesis of 2-Hydroxy-N,N-bis(2-hydroxyethyl)-N-methyl-3-(prop-2-yn-1-yloxy)propan-1-aminium (11)

To a stirring solution of 2,2′-((2-hydroxy-3-(prop-2-yn-1-yloxy)propyl)azanediyl)bis(ethan-1-ol) (10, 0.92 mmol) in acetonitrile (10 mL) was added and K₂CO₃ (0.80 mmol), and the reaction mixture was continued to stir for 30 min at room temperature under N₂ atmosphere. After that, iodomethane (0.92 mmol) was added, and the reaction mixture was heated under reflux condition for 12 h. After maximum consumption of compound 5, the reaction mixture was cooled down to room temperature, and the unused K₂CO₃ was removed by filtration. Removal of organic solvent under reduced pressure yielded a viscous residue. The residue was purified by column chromatography on silica gel using a gradient solvent system of dichloromethane, methanol, and water (5:1:0.1). Characterization of compound 11: ¹H NMR (400 MHz, DMSO-d₆) δ_ppm 4.26 (m, 1H), 4.19 (s, 1H), 3.84 (m, 2H), 3.59 (m, 1H), 3.57 (m, 4H), 3.54 (m, 2H), 3.44 (m, 4H), 3.37 (m, 1H), 3.16 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ_ppm 80.4, 78.1, 72.3, 65.7, 64.9, 64.2, 58.4, 55.4, 50.5; HRMS (ESI) calcd for C₁₁H₂₂NO₄ [M]⁺: 232.1549, found: 232.1554.

Synthesis of tert-Butyl (2-((2-Hydroxy-3-(prop-2-yn-1-yloxy)propyl)amino)ethyl)carbamate (12)

To a stirring solution of compound 6 (1.76 mmol) in dichloromethane (5 mL) was added a solution of tert-butyl (2-aminoethyl)carbamate (3.53 mmol) in dichloromethane at 0 °C under N₂ atmosphere. Then, the reaction mixture was warmed up to room temperature and allowed to stir for 8 h at room temperature. After completion of the reaction, the remaining solvent was removed under reduced pressure. The crude reaction mixture was purified by column chromatography on silica gel using a gradient solvent system of dichloromethane and methanol (10:0.6) to afford compound 12 as a yellow liquid. Characterization of compound 12: yellow liquid (yield 79%) ¹H NMR (600 MHz, CDCl₃) δ_ppm 5.33–5.32 (m, 1H), 4.16 (s, 2H), 3.91–3.88 (m, 1H), 3.55–3.47 (m, 2H), 3.26–3.17 (m, 4H), 2.74–2.72 (m, 2H), 2.65 (t, J = 10.3 Hz, 1H), 2.45 (s, 1H), 1.40 (s, 9H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 156.1, 79.3, 79.1, 74.7, 72.4, 68.4, 58.4, 51.5, 49.0, 39.8, 28.3; HRMS (ESI) calcd for C₁₃H₂₄N₂O₄ [M + H]⁺: 273.1809, found: 273.1827.

Synthesis of N-(2-((tert-Butoxycarbonyl)amino)ethyl)-2-hydroxy-N,N-dimethyl-3-(prop-2-yn-1-yloxy)propan-1-aminium (13)

To a stirring solution of tert-butyl (2-((2-hydroxy-3-(prop-2-yn-1-yloxy)propyl)amino)ethyl)carbamate (12, 0.55 mmol) in acetonitrile (10 mL) was added K₂CO₃ (0.55 mmol), and the reaction mixture was continued to stir for 30 min at room temperature under N₂ atmosphere. After that, iodomethane (1.10 mmol) was added and the reaction mixture was heated under reflux condition for 12 h. After maximum consumption of compound 12, the reaction mixture was cooled down to room temperature, and unused K₂CO₃ was removed by filtration. The removal of organic solvent under reduced pressure yielded a viscous residue. The crude reaction mixture was purified by column chromatography on silica gel using a gradient solvent system of dichloromethane and methanol (10:1) to afford compound 13 as a brown sticky liquid. Characterization of compound 13: brown gummy liquid (yield 92%) ¹H NMR (600 MHz, CDCl₃) δ_ppm 5.85–5.83 (m, 1H), 5.30 (s, 1H), 4.62–4.61 (m, 1H), 4.44 (s, 1H), 4.22 (s, 2H), 3.83–3.81 (m, 2H), 3.69 (m, 4H), 3.45–3.43 (m, 6H), 2.60 (s, 1H), 2.38–2.33 (m, 1H), 1.42 (s, 9H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 156.2, 80.4, 79.1, 75.9, 71.4, 67.0, 64.6, 64.3, 58.8, 53.5, 35.2, 28.4; HRMS (ESI) calcd for C₁₅H₂₉N₂O₄ [M]⁺: 301.2127, found: 301.2133.

Synthesis of N-(2-Aminoethyl)-2-hydroxy-N,N-dimethyl-3-(prop-2-yn-1-yloxy)propan-1-aminium (14)

To a stirring solution of compound 13 (0.465 mmol) in dichloromethane (9 mL) was added trifluoroacetyl (TFA) (1 mL; dropwise addition) at 0 °C and stirring was continued for 30 min. After that, the reaction mixture was warmed up to room temperature and stirring was continued for another 3 h. After the consumption of the maximum starting material, the solvent was removed under reduced pressure. The oily liquid was then washed with diethyl ether (3 × 5 mL). The excess TFA was removed by purging the N₂ gas through the oily liquid yielded as a brown sticky liquid, which was used without further purification. Characterization of compound 14: ¹H NMR (400 MHz, DMSO-d₆) δ_ppm 4.28–4.23 (m, 1H), 4.19 (brs, 2H), 4.12–3.95 (m, 2H), 3.65 (t, J = 7.7 Hz, 2H), 3.49–3.47 (m, 2H), 3.44–3.43 (m, 2H), 3.40–3.36 (m, 1H), 3.44–3.25 (m, 1H) 3.19 (m, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ_ppm 80.3, 78.1, 72.0, 66.9, 64.3, 60.6, 58.4, 52.3, 39.9, 32.9; HRMS (ESI) calcd for C₁₀H₂₁N₂O₂ [M]⁺: 201.1603, found: 201.1617.

General Synthesis of Lipid Molecules^25,41

All lipids were synthesized using the optimized reaction conditions. The H₂O/DMF [1:0.01 (v/v)] was added to a vial containing alkynyl derivative (0.46 mmol), dodecanethiol/hexadecanethiol (1.0 mmol), and TBHP (0.023 mmol) solution and vortexed for 2 min. The vial was sealed and exposed to sunlight (weather condition: sunny; temperature, 30–36 °C). After 30 min, the reaction mixture was diluted with cold water and ethyl acetate. The organic layer was extracted and washed with brine and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure. The reaction mixture was purified by column chromatography using MeOH/CH₂Cl₂ (2–10%) solvent gradient to afford the corresponding lipid.

Characterization of Lipid 15a

Yield 82%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.01–3.96 (m, 4H), 3.7–3.70 (m, 1H), 3.68–3.63 (m, 1H), 3.54–3.45 (m, 2H), 2.97–2.93 (m, 1H), 2.85–2.76 (m, 2H), 2.60–2.52 (m, 4H), 2.40 (s, 6H), 1.59–1.54 (m, 4H), 1.38–1.32 (m, 4H), 1.29–1.24 (m, 37H), 0.87 (t, J = 6.9 Hz, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 73.5, 73.4, 73.1, 73.1, 66.4, 61.8, 61.7, 45.6, 45.4, 34.9, 33.3, 31.9, 31.7, 29.9, 29.7, 29.7, 29.7, 29.7, 29.6, 29.6, 29.4, 29.3–29.2, 29, 28.9, 22.7, 14.2; HRMS (ESI) calcd for C₃₂H₆₇NO₂S₂ [M + H]⁺: 562.4686, found: 562.4664.

Characterization of Lipid 15b

Yield 83%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.92–3.88 (m, 1H), 3.75–3.66 (m, 2H), 3.56–3.45 (m, 2H), 3.00–2.96 (m, 1H), 2.90–2.85 (m, 1H), 2.82–2.78 (m, 1H), 2.62–2.58 (m, 2H), 2.57–2.55 (m, 2H), 2.50–2.43 (m, 1H), 2.32 (s, 6H), 2.30–2.28 (m, 1H), 1.62–1.57 (m, 4H), 1.39–1.37 (m, 4H), 1.27 (s, 54H), 0.89 (t, J = 6.9 Hz, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 73.7, 73.6, 73.2, 66.7, 61.8, 45.6, 34.8, 33.2, 31.9, 31.7, 29.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.3, 29.2, 28.9, 22.7, 14.1; HRMS (ESI) calcd for C₄₀H₈₃NO₂S₂ [M + H]⁺: 674.5938, found: 674.5935.

Characterization of Lipid 16a

Yield 84%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.61–4.56 (m, 1H), 3.89–3.85 (m, 1H), 3.79–3.75 (m, 1H), 3.68–3.60 (m, 1H), 3.58–3.56 (m, 1H), 3.55–3.52 (m, 1H), 3.48–3.45 (m, 9H), 2.95–2.89 (m, 1H), 2.82–2.74 (m, 2H), 2.60–2.50 (m, 4H), 1.59–1.54 (m, 4H), 1.37–1.34 (m, 4H), 1.30–1.22 (m, 36H), 0.87 (t, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 72.7, 72.0, 69.2, 64.6, 64.5, 55.4, 45.5, 35.1, 33.3, 31.9, 29.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.4, 29.3, 29.0, 28.9, 22.7, 14.17; HRMS (ESI) calcd for C₃₃H₇₀NO₂S₂ [M + H]⁺: 577.4921, found: 577.4921.

Characterization of Lipid 16b

Yield 85%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.60–4.53 (m, 1H), 3.87–3.85 (m, 1H), 3.79–3.74 (m, 1H), 3.67–3.59 (m, 1H), 3.58–3.54 (m, 1H), 3.54–3.51 (m, 1H), 3.47 (s, 9H), 2.94–2.89 (m, 1H), 2.81–2.74 (m, 2H), 2.58–2.51 (m, 4H), 1.59–1.53 (m, 4H), 1.37–1.33 (m, 4H), 1.26–1.22 (m, 36H), 0.87 (t, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 72.6, 72.1, 69.2, 65.9, 64.5, 55.4, 45.5, 35.0, 33.3, 31.9, 29.9, 29.7, 29.7, 29.7, 29.7, 29.6, 29.4, 29.3, 29.0, 28.9, 22.7, 14.16; HRMS (ESI) calcd for C₄₁H₈₆NO₂S₂ [M]⁺: 688.6100, found: 688.6091.

Characterization of Lipid 17a

Yield 70%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.65–4.50 (m, 1H), 4.23–4.10 (m, 6H), 3.81–3.62 (m, 2H), 3.56–3.46 (m, 2H), 3.45–3.41 (m, 1H), 3.21–3.16 (m, 1H), 2.97–2.92 (m, 1H), 2.80–2.76 (m, 2H), 2.58–2.52 (m, 4H), 1.59–1.54 (m, 4H), 1.47 (t, 1H), 1.38–1.35 (m, 4H), 1.28–1.25 (m, 35H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 75.7, 73.0, 72.4, 69.5, 67.7, 64.7, 59.1, 46.4, 45.7, 39.5, 35.2, 33.5, 32.2, 30.0, 29.9, 29.9, 29.8, 29.7, 29.6, 29.0, 29.2, 23.0, 14.4; HRMS (ESI) calcd for C₃₄H₇₃N₂O₂S₂ [M]⁺: 605.5113, found: 605.5110.

Characterization of Lipid 17b

Yield 82%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.17–4.68–4.55 (m, 1H), 4.18–4.10 (m, 6H), 3.80–3.57 (m, 2H), 3.48–3.42 (m, 3H), 2.96–2.80 (m, 1H), 2.79–2.74 (m, 2H), 2.63–2.45 (m, 4H), 2.04 (s, 4H), 1.69–1.54 (m, 4H), 1.40–1.33 (m, 4H), 1.30–1.22 (m, 62H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 75.7, 73.0, 72.4, 69.5, 67.7, 64.7, 59.1, 46.4, 45.7, 39.5, 35.2, 33.5, 32.2, 30.0, 29.9, 29.9, 29.8, 29.7, 29.6, 29.0, 29.2, 23.0, 14.4; HRMS (ESI) calcd for C₄₂H₈₉N₂O₂S₂ [M + H]⁺: 717.6365, found: 717.6340.

Characterization of Lipid 18a

Yield 81%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.78 (s, 1H), 4.41 (s, 1H), 4.30–4.28 (m, 2H), 4.13–4.06 (m, 1H), 3.77–3.59 (m, 3H), 3.40 (s), 3.47–3.29 (m, 6H), 2.93–2.88 (m, 1H), 2.82–2.74 (m, 2H), 2.59–2.52 (m, 4H), 1.92–1.85 (m, 1H), 1.59–1.54 (m, 4H), 1.37–1.34 (m, 4H), 1.31–1.20 (m, 38H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 169.4, 72.7, 72.6, 66.8, 64.0, 54.1, 53.1, 45.6, 34.8, 33.1, 31.9, 31.7, 30.0, 29.8, 29.7, 29.7, 29.6, 29.4, 29.1, 29.0, 22.7, 14.1; HRMS (ESI) calcd for C₃₄H₇₀NO₄S₂ [M]⁺: 620.4746, found: 620.4736.

Characterization of Lipid 18b

Yield 83%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.38–4.32 (m, 1H), 4.17–4.07 (m, 1H), 3.82–3.70 (m, 1H), 3.68–3.50 (m, 3H), 3.40–3.32 (m, 6H), 2.94–2.89 (m, 1H), 2.83–2.76 (m, 2H), 2.62–2.53 (m, 4H), 1.61–1.56 (m, 4H), 1.39–1.36 (m, 4H), 1.32–1.22 (m, 51H), 0.91–0.88 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 169.3, 72.6, 72.5, 66.7, 64.0, 54.1, 53.0, 45.0, 34.8, 33.0, 31.8, 31.7, 29.9, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.0, 22.6, 14.0; HRMS (ESI) calcd for C₄₂H₈₆NO₄S₂ [M]⁺: 732.5998, found: 732.5988.

Characterization of Lipid 19a

Yield 81%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.54–4.53 (m, 1H), 4.20–4.15 (m, 4H), 4.05–3.86 (m, 4H), 3.80–3.64 (m, 4H), 3.59–3.52 (m, 2H), 3.43 (s, 3H), 2.97–2.92 (m, 1H), 2.83–2.75 (m, 2H), 2.60–2.53 (m, 4H), 2.04 (s, 1H), 1.63–1.53 (m, 4H), 1.36–1.35 (m, 1H), 1.29–1.23 (m, 38H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 72.6, 72.5, 72.4, 66.0, 65.9, 65.3, 65.2, 56.0, 51.8, 45.4, 34.9, 33.2, 31.9, 31.7, 31.6, 29.9, 29.7, 29.7, 29.6, 29.4, 29.3, 29.0, 28.9, 22.7, 14.1; HRMS (ESI) calcd for C₃₅H₇₄NO₄S₂ [M]⁺: 636.5059, found: 636.5055.

Characterization of Lipid 19b

Yield 85%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 4.55–4.50 (m, 1H), 4.21–4.14 (m, 3H), 3.97–3.86 (m, 4H), 3.78–3.71 (m, 3H), 3.68–3.63 (m, 1H), 3.61–3.50 (m, 1H), 3.43 (s, 3H), 2.96–2.91 (m, 1H), 2.82–2.75 (m, 2H), 2.67–2.51 (m, 4H), 1.87–1.84 (m, 1H), 1.62–1.53 (m, 4H), 1.37–1.34 (m, 4H), 1.30–1.22 (m, 51H), 0.87 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 72.6, 72.5, 72.4, 68.0, 66.1, 65.9, 65.3, 64.5, 56.0, 53.4, 51.8, 46.2, 45.4, 34.9, 33.2, 31.9, 31.7, 31.6, 29.9, 29.7, 29.7, 29.6, 29.4, 29.3, 29.0, 28.9, 25.6, 22.7, 14.1; HRMS (ESI) calcd for C₄₃H₉₀NO₄S₂ [M + H]⁺: 749.6384, found: 749.6382.

Characterization of Control Lipid 20a

Yield 89%. ¹H NMR (600 MHz, CDCl₃) δ_ppm 3.96–3.93 (m, 1H), 3.55 (s, 9H), 3.53–3.49 (m, 1H), 3.31–3.27 (m, 1H), 3.20–3.16 (m, 1H), 3.06–3.03 (m, 1H), 2.96 (s, 1H), 2.88 (s, 1H), 2.77–2.64 (m, 4H), 1.65–1.57 (m, 4H), 1.42–1.63 (m, 4H), 1.30–1.22 (m, 40H), 0.87 (t, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 70.0, 55.7, 46.4, 39.7, 37.4, 33.5, 32.1, 31.7, 29.9, 29.8, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 29.2, 29.0, 22.9, 14.3; HRMS (ESI) calcd for C₃₀H₆₄NS₂ [M]⁺: 502.9685, found: 502.9676.

Preparation of Liposomes

The liposomes were prepared by the thin-film hydration method in Milli-Q water. The synthesized lipids [stock solution of chloroform/methanol at 8:2 (v/v)] were mixed with PE [stock solution of chloroform/methanol at 8:2 (v/v)] with a mole ratio of 1:1 in a glass vial and dried under reduced pressure for 6 h to formulate the lipid thin film. The PE lipid was used as the helper lipid. After that, the thin film was hydrated with Milli-Q water overnight at 55 °C (final concentration of the liposome was 1 mM), and the solution was vortexed for 15 min. Finally, the solution was sonicated for 10 times (30 s of sonication followed by 30 s of cooling on ice) until the thin film disappeared.^42,43

Transmission Electron Microscopy

For transmission electron microscopy (TEM) imaging, vesicles were prepared by the method described above in 25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer, pH 7.4, containing 100 mM KCl.^42,43 The prepared vesicles (no extrusion method was used) were diluted to half of its original concentration using HEPES buffer, pH 7.4. Onto a carbon-coated copper grid, a 10 μL solution of liposome was placed from the diluted solution and allowed to absorb for 1 min. After that, the grid was carefully blotted with filter paper, keeping only a trace amount of the solution in the middle of the grid. Then, the grid was allowed to dry for 10 min at 30 °C. Finally, 5–10 μL of 1% uranyl acetate solution (in water) was added to the grid and allowed to dry for another 1 min. The excess uranyl acetate solution was wicked off, and the grid was dried overnight at 30 °C. The images of the vesicles formed on the carbon-coated copper grid were collected using a JEOL JEM 2100 transmission electron microscope (operated at a maximum accelerating voltage of 200 kV).

Particle Size and Surface Change of the Lipoplexes

The particle size and surface potential of free liposomes and lipoplex (at various N/P ratios) were characterized by using dynamic light scattering (DLS) and ζ potential measurements (Zetasizer Nano ZS90, Malvern, Westborough, MA), respectively, at 25 °C.^42,43

Isoelectric Point Measurements

For the estimation of the isoelectric point (IEP) of the synthesized lipids, the surface potential of lipid was measured in various pH solutions. The liposomes were prepared according to the previously discussed thin-film hydration method, using 5 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.6, containing 5 mM NaCl. For the ζ measurements, isosmotic buffers consisting of 10 mM buffering agent and 10 mM salt at different pH values ranging from 3.0 to 9.0 were freshly prepared. The buffers with the pH range of 3.0–6.5 were prepared using citric acid and trisodium citrate. 3-(N-morpholino)propanesulfonic acid (MOPS) was used for buffer with pH 7.0, and tris(hydroxymethyl)aminomethane (Tris)–HCl was used to prepare buffer with pH values in the range of 7.5–9.0. Disposable capillary cells (DTS1061) and fluorescence cuvettes were used for ζ potential and DLS measurements. All of the analyses were performed at least three times per sample, and the corresponding polydispersity indexes (PDIs) were within 0.1–0.4.

Lipid Phase-Transition Temperature Measurements

To establish the phase-transition temperature (T_m) from the ordered gel to liquid disordered phase of the lipid bilayers, temperature-dependent steady-state anisotropy measurements were performed.^42,43 For this purpose, environment-susceptible fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Vesicles were prepared using a similar method described in the previous section (the hydration was performed by using 20 mM HEPES buffer, pH 7.4, containing 100 mM KCl). A handheld mini-extruder with a polycarbonate membrane (diameter of 100 nm) was used to prepare large unilamellar vesicles. Consequently, to the extruded vesicles (800 μL), 8 μL of 1 mM DPH solution in THF was added (final concentration of DPH was 10 μM; DPH <1% v/v in vesicle solution). Under tumbling condition at room temperature overnight, this liposomal solution was set aside for maximum incorporation of the DPH molecule inside the hydrophobic core of the lipid bilayer. The steady-state fluorescence anisotropy measurements were performed with a refrigerated system using a Peltier temperature controller connected to a Fluoromax-4 spectrofluorometer (Horiba Scientific). At the peak of the fluorescence spectrum, where I_VV and I_VH are the fluorescence intensities of the emitted light polarized parallel and perpendicular to the excited light, respectively, and G = I_VH/I_HH is the instrumental grating factor, the degree of anisotropy (r) in the DPH fluorescence (λ_ex = 350 nm; λ_em = 429 nm) was calculated. All anisotropy values of the DPH probe are the mean values of three individual determinations, before and after the anisotropy measurements [anisotropy (r) = (I_VV – GI_VH)/ (I_VV + 2GI_VH)]. To explore the T_m values of the lipids, plots of the degree of anisotropy (r) of the DPH probe as a function of temperature were studied.

Preparation of Lipoplexes

An assorted amount of liposomes (containing synthesized lipid and PE with a mole ratio of 1:1) was mixed with a constant amount of DNA by pipetting meticulously at a variety of N/P ratios (from 0 to 8), and the mixture was incubated at 30 °C for 30 min to prepare the lipid–DNA complex (lipoplex).^8,10,16,22 The theoretical N/P ratio corresponded to the charge ratio of N atoms on cationic lipid to nucleotide phosphates (in mole) and was calculated by considering the average nucleotide mass.

Gel Retardation Assay

Gel retardation assay is widely used to investigate the formation of liposome/pDNA complex (lipoplex).^8,10,16,22 The stability of lipoplexes of different N/P ratios (ranging from 0 to 8) was measured, as mentioned in the previous section. For this study, a fixed amount of pDNA was used (0.125 μg) and also a fixed amount of each lipoplex solution (20 μL) was electrophoresed on the 0.8% (w/v) agarose gel containing 1 μM ethidium bromide and Tris-acetate (TAE) running buffer at 100 V for 30 min. The pDNA was visualized with a UV lamp using a BioRad Universal Hood. The gel retardation assay in the presence of serum was also performed following similar experimental methods. The lipoplex solution (20 μL; containing 10 or 50% serum) was obtained by adding Tris–HCl buffer and serum (2 or 10 μL) and incubating for a specific time.

Cytotoxicity Assays

The HEK-293T cells were seeded in 96-well plates to get 70% confluency after 12 h incubation at 37 °C. Different concentrations of the compound were prepared in plain Dulbecco’s modified Eagle’s medium (DMEM) and added to the cells directly after a phosphate-buffered saline (PBS) wash. The MTT solution was prepared at 5 mg/mL in PBS and filtered through a 0.2 μm filter. After 24 h, 100 μL of 10% diluted MTT in plain DMEM was added in each well. Cells were incubated for 4 h at 37 °C with 5% CO₂, 95% air, and complete humidity. After 4 h, the MTT solution was removed and replaced with 100 μL of DMSO. The plate was further incubated for 15 min in the dark at room temperature. The optical density (OD) of the wells was determined using a Multiscan Go plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm.

Measurement of Transfection Efficiency

To check the transfection efficiency of the potent lipids, we used HEK-293T cells. The HEK-293T cells were seeded in 12-well plates with a density of 0.1 × 10⁶ per well, containing DMEM and 10% fetal bovine serum (FBS), and the plates were incubated for 12 h at 37 °C. For the transfection experiment, first, pMMP7-GFP plasmid (1.5 μg) and Opti-MEM media were added in a microfuge tube, and the final volume was adjusted to 150 μL. In another centrifuge tube, lipoplex of different N/P ratios and Opti-MEM media were added, and the final volume was adjusted to 150 μL. Then, the microfuge tubes were incubated for 5 min at 37 °C. After that, the solutions were mixed and kept at room temperature for 30 min. Before transfection, the media of 12-well plates was removed and the cells were washed with PBS. Then, the media containing lipoplex (300 μL) was added in each well and the cells were incubated for 4 h at 37 °C. After that, the medium was replaced with fresh DMEM with 2% FBS and incubated for another 24 h. Finally, the transfected cells were analyzed to measure GFP fluorescence-labeled cells after 24 h, under 40× magnifications with a green filter using Floid imaging system. Lipofectamine 2000 was used as the positive control to measure the transfection efficiency. For all of the measurements, 0.2 μL of Lipofectamine 2000 from the commercially available stock was used as control (as no concentration was provided by the commercial source).

GFP-Uptake Assays

The HEK-293T cells were seeded in six-well plates (density 0.3 × 10⁶ per well in DMEM containing 10% FBS). The plates were then incubated overnight at 37 °C. For transfection, the pMMP7-GFP (2.0 μg) and Opti-MEM solution were added to make the final volume of 150 μL in a centrifuge tube. In a separate tube, the lipoplex (at different N/P ratios) and Opti-MEM media (to make the final volume of 150 μL) were added. Then, both the tubes were incubated for 5 min at room temperature. The solutions were mixed and kept at room temperature for 30 min. The wells of the plate were washed with PBS. Then, media containing lipoplex (300 μL) were added in each well and the cells were incubated for 4 h at 37 °C. After that, the medium was replaced with fresh DMEM with 2% FBS and incubated for another 24 h. For FACS analyses, experiments were performed on BD FACSCalibur using the FL1 channel for green fluorescence. Data analyses were done through FCS Express 6 Flow Cytometry software. Autofluorescence was detected using nontransfected cells as control, indicated as a peak in the histogram plot using FL1 channel. The M1 region is shown in plots capturing green fluorescence intensity in transfected cells, which is higher than autofluorescence intensity. Approximately 10 000 events were statistically evaluated for each sample in the histogram.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03413.

• Experimental details of biophysical studies, MTT assay, and cellular uptake assay (PDF)

Author Contributions

S.D. performed the synthesis and biophysical measurements. A.S. and S.P. performed biophysical measurements and data analysis. A.G. and S.K. designed and performed the cellular studies. D.M. designed and coordinated the overall project. S.D. and D.M. wrote the manuscript.

The authors gratefully acknowledge the Department of Biotechnology, Govt. of India (MED/2015/04), and Science and Engineering Research Board, Govt. of India (EMR/2016/005008), for the financial support.

The authors declare no competing financial interest.