Sulfonium-based liposome-encapsulated antibiotics deliver a synergistic antibacterial activity

Abstract

The devastating antibacterial infections, coupled with their antibiotic resistance abilities, emphasize the need for effective antibacterial therapeutics. In this prospect, liposomal delivery systems have been employed in improving the efficacy of the antibacterial agents. The liposome-based antibiotics enhance the therapeutic potential of the new or existing antibiotics and reduce their adverse effects. The current study describes the development of sulfonium-based antibacterial lipids that demonstrate the delivery of existing antibiotics. The presence of cationic sulfonium moieties and inherent membrane targeting abilities of the lipids could help reduce the antibiotic resistance abilities of the bacteria and deliver the antibiotics to remove the infectious pathogens electively. The transmission electron microscopic images and dynamic light scattering analyses revealed the liposome formation abilities of the sulfonium-based amphiphilic compounds in the aqueous medium. The effectiveness of the compounds was tested against the Gram-negative and Gram-positive bacterial strains. The viability of the bacterial cells was remarkably reduced in the presence of the compounds. The sulfonium-based compounds with pyridinium moiety and long hydrocarbon chains showed the most potent antibacterial activities among the tested compounds. Mechanistic studies revealed the membrane-targeted bactericidal activities of the compounds. The potent compound also showed tetracycline and amoxicillin encapsulation and sustained release profiles in the physiologically relevant medium. The tetracycline and amoxicillin-encapsulated lipid showed much higher antibacterial activities than the free antibiotics at similar concentrations, emphasizing the usefulness of the synergistic effect of sulfonium-based lipid and the antibiotics, signifying that the sulfonium lipid penetrated the bacterial membrane and increased the cellular uptake of the antibiotics. The potent lipid also showed therapeutic potential, as it is less toxic to mammalian cells (like HeLa and HaCaT cells) at concentrations higher than their minimum inhibitory concentration values against S. aureus, E. coli, and MRSA. Hence, the sulfonium-based lipid exemplifies a promising framework for assimilating various warheads, and provides a potent antibacterial material.

The devastating antibacterial infections, coupled with their antibiotic resistance abilities, emphasize the need for effective antibacterial therapeutics.

Introduction

Antimicrobial drug resistance has intimidated the success of preventing and treating infectious diseases, and other ailments such as cancer.^1,2 Various medical procedures, such as major surgery and organ implant, are also facing uncertain outcomes due to bacterial infection and antibacterial drug resistance.^1–4 Over the years, bacteria such as Escherichia coli (E. coli), Klebsiella pneumoniae, and Staphylococcus aureus (S. aureus) have developed multidrug resistance, which is challenging to treat with common antibacterial drugs. The healthcare encumbrance related to the drug-resistant bacteria is further impaired due to the ability of several bacterial strains to become resistant towards the frontier antibiotics, such as vancomycin. Overexposure to drugs allows the bacteria to develop drug resistance capability by modifying their genetic material. A plethora of bacteria also produce a sturdy biofilm to cover their colony and protect themselves from unpropitious conditions.^5,6 Thus, new or modified antibacterial drugs are highly demanding. Unexpectedly, the span is too large between the rate of development of new medicines and the evolution of another drug-resistant bacterial strain. Hence, exploring both new antibacterial agents and their delivery systems continues to be an imperative research emphasis, as it is crucial to fight against the emerging apprehension of drug-resistant pathogenic bacteria. A combinatorial strategy could also help to fight against bacteria, as the synergistic effect is likely to demonstrate the superior bactericidal effect, reduce the host-specific toxicity, and operational killing. The therapeutic efficiency of this antibacterial strategy depends on the ability of the carrier to infringe the intrinsic resilience of the bacterial cells, and in re-establishing susceptibility to the antibacterial agent.⁷

Antibacterial drugs primarily act on the bacterial cell wall, plasma membrane, replication machinery, metabolic activity, and protein translation machinery. Drugs with membrane-targeted activity have an important role in the antibacterial activity, and comparatively, they can better counteract the bacterial resistance system. The membrane-targeted antibacterial drugs provide the extra benefit without developing resistance as they can act effectively towards the dormant or spore form of the bacteria and biofilm-forming strain because of the conserved membrane composition in all growth forms of the bacterial cell and distinct mechanism of action that implicates less specific membrane permeabilization.^8–10 Membrane targeting antibacterial agents generally leads to bactericidal activity. Various reports have already demonstrated that bactericidal drugs are more preferred over bacteriostatic drugs to treat meningitis, endocarditis, and several other bacteria-related diseases.^11,12 Membrane targeting antibacterial drugs target the bacterial cell membrane over mammalian cells because of the differences in the lipid component and composition of their architecture.¹³

In this regard, small molecule-based membrane-active synthetic amphiphilic compounds are considered as potential antibacterial agents that hold the prospect of success for combinatorial antimicrobial therapy. Amphiphilic compounds with a cationic moiety convey membrane-oriented activity because of the negatively charged lipopolysaccharide (LPS) and teichoic acid (TA) in Gram-negative and Gram-positive bacteria, respectively.^14–17 The hydrophobicity of an antimicrobial compound can easily coordinate the bacterial membrane and is the panacea of water-soluble antibiotic resistance because of the reduced porins in the bacterial outer membrane. Furthermore, hydrophobic antimicrobial ointments are becoming the choice of wound dressings to combat the severe skin infection caused by superbugs, like methicillin-resistant S. aureus (MRSA). However, the therapeutic prospective of synthetic amphiphilic compounds depends on their host-specific toxicity, suggesting the requirement of the judicious design of antibacterial amphiphilic compounds so that both carrier and antibiotics are accessible to the bacterial cells to achieve the synergistic effect. Different research groups have demonstrated that the cationic sulfonium-based compounds are potent antimicrobial agents, and less toxic than the quaternary ammonium and phosphonium-based compounds and other cationic antimicrobial agents.^15,16,18 Sulfonium-based compounds are naturally abundant in plants and animals. Methyl sulfonium-containing compounds are commonly used as a methyl transfer agent.¹⁹ However, the antibacterial activities of these sulfonium-based compounds are rarely studied, and practically used in comparison to that of ammonium and phosphonium-based compounds. Currently, few sulfonium-based compounds, such as adenosylmethionine, bleomycin, and modified-echinomycin, are used in therapeutics. Recent studies revealed that the sulfonium-linked vancomycin analog had enhanced antibacterial activity against vancomycin-resistant bacteria both under in vitro and in vivo conditions. The cationic sulfonium moiety improved the interaction of vancomycin with the cell membrane and disturbed the integrity of the membrane of both Gram-negative and Gram-positive bacteria.¹⁸ In our previous study, we demonstrated that the sulfonium lipids encapsulate and deliver the anticancer drug, doxorubicin, to a mammalian cell.²⁰ The self-assembly properties of those sulfonium lipids could be utilized to deliver both aqueous soluble and insoluble antibiotics at the target site. The amphiphilicity and the presence of cationic sulfonium moieties would also allow the sulfonium-based lipids to fuse with the outer membrane of the bacterial cells and release the encapsulated antibiotics.²¹ Recently, polymeric amphiphilic systems with inherent antibacterial activity were reported.²² However, a small molecule-based biocompatible amphiphilic system can augment the therapeutic benefits.

Herein, we report the synthesis and mechanism of the antibacterial activity of sulfonium-based compounds. The role of the cationic charge and hydrocarbon chain length in antimicrobial activity was investigated against both Gram-positive and Gram-negative strains of bacteria. The amphiphilic molecule with the pyridinium headgroup showed the most potent antibacterial activity among the tested compounds against Gram-negative, Gram-positive, and even drug-resistant bacteria. The less-toxic sulfonium compound could also prevent the formation of a biofilm. In addition, encapsulation of the commercial antibiotics and antibacterial efficacies of the composite was investigated. The composite showed moderate loading of water-soluble antibiotics and a sustained release profile. Overall, our studies propose that the electrostatic interaction of cationic liposomes with the bacterial membrane allows its disassembly and insertion of the amphiphilic compound to the bacterial membrane, resulting in the concomitant release of antibiotics at the target site to achieve synergistic antibacterial activities.

Results and discussion

Design and synthesis of compounds

The anticancer drug delivery and moderate antimicrobial activities of the sulfonium lipids motivated us to synthesize a new series of sulfonium-based compounds.²⁰ The key features in designing the compounds were the installation of a cationic (pyridinium) or anionic (sulfonic acid) headgroup and variation of the alkyl chain length, in addition to the sulfonium moieties (Scheme 1). The variation in the alkyl chain length allowed us to investigate the role of hydrophobicity and antimicrobial activity. We envisage that the hydrophobicity of the dialkyl chain lengths could enable the compounds to self-aggregate in an aqueous medium, which could be useful in encapsulating the commercial antibiotics. The presence of both sulfonium moieties and antibiotic encapsulation ability would generate a composite antibacterial agent, which can increase the bactericidal effect, reduce the antibiotic-related toxicity, and enhance the efficacy of the antibiotics at lower doses (Fig. 1A). Most of the conventional liposomes, micelles, or nanoparticle-based delivery systems lack such a dual mode of action, suggesting that the combined antibacterial activity of these sulfonium-based compounds can improve the killings of drug-resistant pathogenic bacteria.

Scheme 1. Synthetic routes to the sulfonium-based compounds.

Fig. 1. Cartoon diagram demonstrating the probable pathway for the bactericidal effect of the antibiotics-encapsulated liposomes of compound 7a (A). Representative TEM image of the soluble aggregates generated from the 100% compound 7a (B). The scale bar for the TEM image is 200 nm. Variation of the hydrodynamic diameter of the soluble aggregates generated from the 100% compound 7a at different pH values was measured by DLS analysis (C). The surface potential of the soluble aggregates generated from the 100% compound 7a at different pH values (D).

The compounds were synthesized according to our reported methods with minor modifications (Scheme 1).²⁰ The compounds for the current study were synthesized using 1,3,5-tris(bromomethyl)benzene. The mono-modification of 1,3,5-tris(bromomethyl)benzene with azide and pyridine resulted in compounds 2 and 3, which were further modified with aliphatic thiols to provide compounds 4 and 5, respectively. The azide–alkyne click reaction of compound 4 with prop-2-yne-1-sulfonic acid produced compound 6. Finally, the treatment of compounds 5 and 6 with methyl iodide in the presence of AgBF₄ provided the desired products 7 and 8 with satisfactory yield. These compounds were characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS).

Aggregation behavior in aqueous solution

To investigate the behavior of these amphiphilic compounds under an aqueous environment, the compounds were dissolved in phosphate-buffered saline (PBS).²⁰ Compound 7b was completely soluble, but compounds 7a and 8 formed a suspended aqueous solution. The presence of a short alkyl chain length and sulfonic acid could be reasons for their aqueous solubility. The field-emission transmission electron microscope (FE-TEM) analysis showed that compound 7a forms spherical aggregates in an aqueous medium (Fig. 1B). The dynamic light scattering (DLS) study showed that the size of the spherical aggregates varies between 270–340 nm at different pH values of the respective buffers at 25 °C (Fig. 1C and D, and S6†). The zeta-potential measurements showed that the overall surface charge of the spherical aggregates was positive, and the positive charge increases with the decrease in pH of the buffer, which could be due to the reduction in the solvation number in the acidic medium. Hence, TEM, DLS, and zeta-potential measurements revealed that compound 7a formed stable spherical aggregates with positive surface potential.

Antibacterial activities of the compounds

The sulfonium-based compounds showed antibacterial activity against both Gram-positive and Gram-negative bacteria.^18,20 The minimum inhibitory concentrations (MIC) of the compounds were calculated against Gram-negative bacteria, such as E. coli MTCC 1687 and P. aeruginosa MTCC 2488, and Gram-positive bacteria, such as S. aureus MTCC 96 and methicillin-resistant S. aureus (MRSA), by micro broth dilution method.²³ The highly infectious E. coli strain resides in the intestine and causes various diseases, including pneumonia, diarrhea, and urinary tract infection (UTI). The bacterial strains of S. aureus and Pseudomonas aeruginosa (P. aeruginosa) are the frequent residents of bronchioles and alveoli of cystic fibrosis patients. The MIC values were reckoned as the minimum concentration of the compounds, which caused no visible growth, or the optical densities (OD) of the compound-treated bacterial cultures were close to that of the control (without any bacteria). Compounds 7a and 8 showed very good antibacterial activity against all four tested bacterial strains (Table 1). The MIC values of the pyridinium-containing compound 7a were within 6.3–12 μM, whereas the sulfonic acid-containing compound 8 had slightly higher MIC values of 12–24 μM. It was encouraging to observe that the drug-resistant strain of S. aureus (MRSA) showed a similar MIC value to that of its drug-sensitive strain. Further antibacterial studies of compounds 7a and 8 were performed to investigate the impetus of different structural moieties in the compound, which provide the observed antimicrobial property. However, compound 4 with azide but no sulfonium moieties showed no antibacterial activity, even at 100 μM.²⁰ Compound 5a, with only pyridinium but no sulfonium moieties, showed no antibacterial activity even at 100 μM, suggesting the role of the sulfonium moieties in the antibacterial activities of compound 7a. Compound 7b, with the same headgroup but with a short alkyl chain (ethyl group) length compared to that of compound 7a, showed no antibacterial activity even at 100 μM, suggesting that the long hydrocarbon chain is as important as other moieties in the compound. Compound 5b with a short alkyl chain length also showed no antibacterial activity. The sulfonium and pyridinium or sulfonic acid moieties could probably be involved in electrostatic or hydrogen bond interactions with the phosphoryl and carboxyl groups of the bacterial membrane lipids, and elicit the insertion of the long hydrocarbon chain lengths of compounds 7a and 8 to the bacterial membrane.¹⁸ The higher antibacterial activity of compound 7a over 8 could be due to much stronger electrostatic interaction of the pyridinium moiety with the anionic lipids of the bacterial membrane compared to that of the sulfonic acid moiety. The cationic moieties showed better antibacterial activity over neutral or anionic moieties.^14–16 Similar antibacterial activities were reported when the sulfonium moiety was appended to vancomycin.¹⁸ The sulfonium-containing vancomycin analogue showed improved antibacterial activity against vancomycin-resistant bacteria, suggesting the potential of the sulfonium moiety in antibacterial activities. The attachment of the sulfonium moiety also alters the innate feature of vancomycin, leading to its activity against Gram-negative bacteria.¹⁸ Interestingly, the antibacterial activity of these sulfonium lipids 7a and 8 are similar to other substances, especially to some widely used antiseptics. The MIC values of those antiseptics against P. aeruginosa are the following: for octenidine dihydrochloride, 3.9–31.3 μg mL⁻¹ (6.25–50.1 μM); for chlorhexidine digluconate, 15.6–64.0 μg mL⁻¹ (30.9–126.6 μM); and for polyhexamethylene biguanide, 2–31.3 μg mL⁻¹.^24,25 Simultaneously, the activity of sulfonium compounds 7a and 8 against P. aeruginosa is significantly higher than other very important antiseptics, like benzalkonium chloride (MICs, 32–512 μg ml⁻¹), povidone-iodine (MICs, 62.5–1024 μg ml⁻¹), and triclosan (MICs > 512 μg ml⁻¹).^24,25 Several sulfonium-based compounds showed better antimicrobial activities than some common cationic antimicrobials, including cetylpyridinium chloride and benzalkonium chloride.²⁶ The recently developed sulfonium N-chloramines showed higher antibacterial activities than the formerly reported quaternary ammonium counterpart.²⁷ The polymeric salts of p-vinylbenzyl tetramethylenesulfonium tetrafluoroborate exhibited high antibacterial activity against Gram-positive bacteria.²⁸

Antibacterial activities of the synthesized compounds.

Compound			MIC (μM)/MBC (μM)				HC50 (μM)
	n	R	E. coli	P. aeruginosa	S. aureus	S. aureus (MRSA)
4	11	–N3	>100	>100	>100	>100	—
5a	11		>100	>100	>100	>100	—
5b	2		>100	>100	>100	>100	—
6	11		>100	>100	>100	>100	—
7a	11		7.5 ± 1/10 ± 1	12 ± 0.5/24 ± 1	6.3 ± 1/12.5 ± 1	6.3 ± 1/12.5 ± 1.5	29
7b	2		>100	>100	>100	>100	—
8	11		12 ± 1/20 ± 1	25 ± 1/50 ± 1	10 ± 0.5/12.5 ± 1	25 ± 1/>100	35
Tetracycline			8.5 ± 1	14 ± 0.5	4.2 ± 0.5	>100
Amoxicillin			11 ± 1	4.3 ± 0.5	6.8 ± 1	>50

The bactericidal activities of the compounds were tested against the same bacterial strains.²⁹ The minimum bactericidal concentrations (MBC) of the compound were higher than the MIC values (bacteriostatic concentration),³⁰ suggesting that a higher concentration of the compound is required to demolish the bacterial membrane or metabolic activity at a rate with which they might not be able to repair it and die off (Table 1). Thus, these amphiphiles are proficient in killing the bacterial cell at a low concentration, making them good antimicrobial agents. The bactericidal activity of the compounds was further confirmed by FESEM analysis (Fig. 2).²⁰ A significant difference in morphology of the bacterial cells was observed between the control and compound-treated samples. The cocci shape of the S. aureus and the rod shape of E. coli were disfigured or fragmented in the drug-treated bacterial sample, which could be due to the relentless disruption of the bacterial membrane. These morphological changes of the bacterial cells suggest that compound 7a may induce bacterial cell death.^17,20 Additionally, the antibiofilm activity of the compound was tested. The extra polymeric substance or biofilm is essentially composed of extracellular DNA (eDNA), peptide or protein, and intercellular polysaccharide with different ratios and architecture, depending upon the bacterial strain. The biofilm has been implicated in many infectious diseases, including chronic sinusitis, atherosclerosis, chronic wounds, endocarditis, and many more.³¹ The antibiofilm assay by the crystal violet staining method showed that compound 7a inhibits the formation of a biofilm by the S. aureus strain in a dose-dependent manner (Fig. 3A).³² However, the concentration of 7a required (24 μM) to abolish mature biofilm was 2–4-fold higher than the concentrations necessary to inhibit the formation of 90% biofilm (12 μM) (Fig. 3B and C). Thus, these amphiphiles have the capability to degrade the mature biofilm. The presence of both pyridinium and sulfonium (cationic) moieties could assist the interaction of compound 7a with the TA and LPS of the Gram-positive and Gram-negative bacterial cells. This electrostatic interaction could drive the membrane-mediated antibacterial activities of compound 7a (Fig. 3C). The mechanism of the bactericidal activity was studied by propidium iodide (PI) uptake assay.^33,34 The fluorescent DNA intercalating agent, PI can cross the damaged cell membrane, but is impermeable to the intact and live cell. The PI uptake assay was performed at different concentrations and time intervals using S. aureus cells. The increase in the fluorescence intensity of PI revealed that the antibacterial activity of compound 7a is membrane-directed and time-dependent, suggesting that the number of dead cells increases with time (Fig. 4A). Further mechanistic studies were performed by membrane depolarization assay using the DiSC₃(5) dye.³⁴ The fluorescence intensity of this dye gets quenched on the negatively charged bacterial membrane. However, it showed higher fluorescence intensity on the disrupted membrane. The DiSC₃(5) assay was tested on S. aureus at various concentrations of compound 7a and positive control (valinomycin). A significant disparity was observed in the fluorescence intensity even at a very low concentration range, increasing with the increase in concentration. The increase in the fluorescence intensity of the DiSC₃(5) dye of the compound 7a-treated cells suggests the disruption in the membrane integrity of S. aureus cells (Fig. 4B). A fluorescence-based live and dead cell imaging study by staining the drug-treated cells with PI and 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA-SE) was also done to investigate the bactericidal activity of the compound (Fig. 4C). The membrane-permeable nonfluorescent cFDA-SE dye only shows fluorescence due to the formation of carboxyfluorescein succinimidyl ester (CFSE) in the presence of esterase enzyme, which is an indicator of bacterial cell viability. During the measurements, the S. aureus cells were stained with the cFDA-SE dye, and microscopic fluorescence images were recorded. The analysis revealed that the compound 7a-treated S. aureus cells lacked CFSE fluorescence, which demonstrated the inactivity of bacterial metabolism due to superior membrane damage, which resulted in cell death.

Fig. 2. Representative FESEM images of untreated (A and C) and compound 7a-treated (B and D) bacterial cells. The scale bars for the images are 1.0 μm (A, C, and D) and 200 nm (B).

Fig. 3. Compound 7a-mediated eradication of the S. aureus (MRSA) biofilm was investigated by crystal violet staining assay (A and B). The diagram demonstrates the probable interaction pattern of compound 7a with the negatively charged TA and LPS in the Gram-positive and Gram-negative bacteria, respectively (C).

Fig. 4. Fluorescence-based PI-uptake assay of compound 7a using S. aureus (MRSA) cells (A). Compound 7a-treated membrane depolarization assay using DiSC₃(5) dye on S. aureus (MRSA) cells (B). Representative fluorescence microscopic images of untreated and compound 7a-treated bacterial cells (C). The scale bar for the images is 10 μm.

Antibiotics encapsulation and antibacterial efficacy

The self-assembly property of compound 7a inspired us to encapsulate the antibiotics and generate an antibacterial composite, where antibiotic and sulfonium-based compounds would show a potential synergistic effect. When encapsulated in liposomes, antibiotics and other drugs are more effective and protected from the adverse environment of the cell.³⁵ To explore the antibiotic encapsulation, both bacteriostatic and bactericidal antibiotics tetracycline and amoxicillin, respectively, were selected. Encapsulation and release profiles of the antibiotics in compound 7a were investigated by the fluorometric method (Fig. S7 and S8†).³⁶ Compound 7a showed the satisfactory loading ability for tetracycline (18%) and amoxicillin (28%) at pH 7.4, suggesting that these water-soluble antibiotics could be entrapped within the hydrophilic core of the self-assembled structure (Fig. 5A). Fluorescence measurements of these antibiotics showed sustained release profiles with 40% and 60% of tetracycline and amoxicillin, respectively, which were released after 60 hours of incubation at 37 °C (Fig. 5B). These sustained antibiotics release profiles of compound 7a are advantageous for their biological applications, including a reduction in antibiotic-associated toxicity, augmentation of the activity of the antibiotics at lower doses, and others.

Fig. 5. Cartoon diagram demonstrating the antibiotic release pathways from the liposomes of compound 7a (A). The antibiotics (tetracycline and amoxicillin) release profile of compound 7a at pH 7.4 (B). Concentration-dependent viabilities of the HeLa and HaCaT cells in the presence of compounds 7a and 8 (C). Concentration-dependent viabilities of the HaCaT cells in the presence of compounds 7a and antibiotics-encapsulated compound 7a (D).

The antibacterial activity of tetracycline and amoxicillin encapsulated liposomes of 7a (tetracycline@7a and amoxicillin@7a) showed synergistic effects (Table S1†), and the antibacterial activities were 4 to 6.5-folds lower than that for free tetracycline and amoxicillin, suggesting the accomplishment of dual activity of compound 7a and antibiotics encapsulated within the liposome (Table 2). Therefore, the encapsulation of these antibiotics increases the drug efficacy, and the controlled release of drugs could provide the long-term effect and minimize the necessity of multiple dosing.

Antibacterial activities of the commercial antibiotics and antibiotic-encapsulated sulfonium-based amphiphilic compound.

Compound	Bacterial train	MIC (μM)	MIC (antibiotic@7a; μM)	MIC of 7a in antibiotic@7a (μM)
7a	S. aureus	6.3	—	—
	E. coli	7.5	—	—
Tetracycline	S. aureus	4.2	0.7	1.0
	E. coli	8.4	2.0	1.4
Amoxicillin	S. aureus	6.8	1.2	1.0
	E. coli	10.9	1.7	1.3

An antimicrobial agent can be used for further bio-application only when it is biocompatible and non-toxic to human cells. As our drug targets the bacterial membrane, it should be specific to that only. Although the bacterial membrane differs from the mammalian cell membrane and red blood cell membrane architecture, the drug should be tested against mammalian cells because of the few similarities in the membrane nature. In this regard, the cytocompatibility of the synthesized potent amphiphiles and antibiotic-loaded amphiphiles were verified against HeLa (human cervical cancer) and HaCaT (human keratinocyte; normal cells) cells by assessing the NADH-dependent cellular oxidoreductase enzyme activities with the MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Fig. 5C). The HaCaT cell line showed no toxicity up to 100 μM against compound 8. The calculated IC₅₀ value of compound 7a was 27 μM, while the IC₅₀ values of tetracycline@7a and amoxicillin@7a were 8 and 29.41 μM, respectively (Fig. 5D). Furthermore, the toxicity of the synthesized amphiphiles was tested against the membrane of red blood cells, which is very much elastic, and the only structural component it has is the plasma membrane that provides mechanical support to the cell. To ensure the compatibility of the sulfonium-based amphiphiles against red blood cells, the haemolytic activity was performed. Disruption of the membrane of the red blood cells causes heme leakage from the cell that can be measured by the spectrophotometric method (Fig. S9†). Amphiphiles at various concentration ranges were incubated with 5% haematocrit at room temperature. The extent of heme release was measured by UV-vis spectroscopy at 410 nm after centrifugation. The result was compared with negative (no compound) and positive controls (0.1% Triton X-100), and it was found that at the MIC and MBC value, the compound has very less or no haemolytic activity (Table 1 and Fig. S9†). The calculated therapeutic index (TI; IC₅₀/MIC) of compound 7a and the composites were within 3.6–29.4 for both E. coli and S. aureus bacterial cells (Table S2†).

Experimental

I. Synthesis and characterization of the compounds

Synthesis of 1-(azidomethyl)-3,5-bis(bromomethyl)benzene (2)

To a stirring solution of 1,3,5-tris(bromomethyl)benzene (1) (1.40 mmol) in dry DMF solvent, sodium azide (1.2 mmol) was added portion-wise, and the resulting mixture was stirred for 3 hours at room temperature. The progress of the reaction was monitored by thin-layer chromatography (TLC). After the maximum consumption of compound 1, the solvent was removed under reduced pressure, and CH₂Cl₂ (25 mL) was added. The salt was filtered off, and the filtrate was concentrated under reduced pressure. The crude product was purified through silica gel column chromatography with a solvent gradient system using ethyl acetate and hexane (0–1.5% EtOAc in hexanes) to obtain the pure product 2 as a clear yellow oil (200 mg; 45% yield). The compound was characterized by ¹H and ¹³C NMR, and the results were in accordance with the literature report.³⁷

Synthesis of 1-(3,5-bis(bromomethyl)benzyl)pyridin-1-ium (3)

To a stirring solution of 1,3,5-tris(bromomethyl)benzene (1, 1.40 mmol) in acetone solvent, pyridine (1.3 mmol) was added. The resulting mixture was stirred for 12 hours at room temperature. A white precipitate was observed, which was filtered and washed with acetone to afford the desired product 3 as a white solid (350 mg, 70%). The compound was characterized by ¹H and ¹³C NMR, and the results were in accordance with the literature report.³⁸

Synthesis of ((5-(azidomethyl)-1,3-phenylene)bis(methylene))bis(dodecylsulfane) (4)

To a stirring solution of 1-(azidomethyl)-3,5-bis(bromomethyl)benzene (2, 0.31 mmol) in CH₃CN/H₂O (3 : 1 in volume) was slowly added (dropwise) a previously stirring solution of dodecanthiol and sodium bicarbonate in CH₃CN/H₂O (3 : 1 in volume) at room temperature. The resulting reaction mixture was stirred for 36 hours. After that, the solvent was removed under reduced pressure. Then, the reaction mixture was diluted with cold water and ethyl acetate. The organic layer was extracted with EtOAc (3 × 50 mL) and washed with brine, and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure. The crude reaction mixture was purified through silica gel column chromatography with a solvent gradient system using ethyl acetate and hexane (0–10% EtOAc in hexane) to afford compound 4 as a colorless gummy solid. Characterization of the compound: colourless gummy liquid (yield – 87%) ¹H NMR (600 MHz, CDCl₃) δ_ppm 7.23–7.22 (m, 1H), 7.14 (s, 1H), 4.31 (s, 2H), 3.69–3.66 (m, 4H), 2.42–2.37 (m, 4H), 1.57–1.50 (m, 4H), 1.35–1.24 (m, 36H), 0.88 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 139.7, 135.8, 129.3, 127.2, 54.5, 36.0, 31.9, 31.4, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9, 22.7, 14.1; HRMS (ESI) calcd. for C₃₃H₆₃N₄S₂ [M + NH₄]⁺: 579.4494, found: 579.4463.

Synthesis of 1-(3,5-bis((dodecylthio)methyl)benzyl)pyridin-1-ium (5a)

To a stirring solution of 1-(3,5-bis(bromomethyl)benzyl)pyridin-1-ium (3, 0.14 mmol) in CH₃CN/H₂O (3 : 1 in volume) was slowly added (dropwise) a previously stirring solution of dodecanthiol (0.30 mmol) and sodium bicarbonate (0.30 mmol) in CH₃CN/H₂O (3 : 1 in volume) at room temperature. The resulting reaction mixture was stirred for 36 hours. After that, the solvent was removed under reduced pressure. Then, the reaction mixture was diluted with cold water and ethyl acetate. The organic layer was extracted with EtOAc (3 × 50 mL) and washed with brine, and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure. The crude reaction mixture was purified through silica gel column chromatography with a solvent gradient system using ethyl acetate and hexane (0.1–10% EtOAc in hexanes) to afford compound 5a as a colorless gummy liquid. Characterization of the compound: colorless gummy liquid (yield – 81%) ¹H NMR (600 MHz, CDCl₃ + DMSO-d₆) 9.38–9.37 (m, 2H), 8.38–8.34 (m, H), 7.99–7.95 (m, 1H), 7.33 (s, 1H), 7.27 (s, 1H), 7.20 (s, 1H), 6.17 (s, 2H), 2.68 (s, 1H), 2.37–2.30 (m, 4H), 1.51–1.43 (m, 4H), 1.25–1.18 (m, 38H), 0.81 (t, 3H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 145.2, 145.0, 140.9, 132.9, 130.9, 128.3, 128.2, 64.3, 41.0, 36.0, 32.1, 31.9, 29.6, 29.5, 29.3, 29.2, 28.9, 22.6, 14.0; HRMS (ESI) calcd. for C₃₈H₆₄NS₂ [M]⁺: 598.4480, found: 598.4487.

Synthesis of ((5-(pyridin-1-ium-1-ylmethyl)-1,3-phenylene)bis(methylene))bis(dodecyl(methyl)sulfonium) (7a)

To a stirring solution of 1-(3,5-bis((dodecylthio)methyl)benzyl)pyridin-1-ium (5a, 0.08 mmol) in dry DCM solvent was added AgBF₄ (0.08 mmol) and methyl iodide (0.2 mmol), and the resulting mixture was stirred for 5 hours at room temperature. The progress of the reaction was monitored by TLC. After consumption of compound 5a, the solvent was removed under reduced pressure and the resulting mixture was filtered off using a pad of celite to remove silver salt. Then, the crude mixture was washed with ether and CH₃CN to afford compound 7a as a brown gummy liquid. Characterization of the compound: brown gummy liquid (yield – 77%); ¹H NMR (600 MHz, DMSO-d₆) δ_ppm 9.14–9.13 (m, 2H), 8.70–8.67 (m, 1H), 8.24–8.21 (m, 2H), 7.63 (s, 2H), 7.57 (s, 1H), 5.93 (s, 1H), 4.80–4.63 (m, 4H), 2.79 (s, 6H), 1.76–1.63 (m, 4H), 1.34–1.18 (m, 39H), 0.86 (t, 6H); ¹³C NMR (151 MHz, DMSO-d₆) δ_ppm 146.8, 145.5, 136.6, 132.4, 130.9, 128.9, 62.9, 46.1, 44.1, 40.91, 31.7, 29.5, 29.4, 29.2, 29.1, 28.8, 28.2, 23.7, 22.5, 21.9, 14.4; ES-MS (ESI+) m/z: [(M + 3BF₄⁻] 609.4756.

Synthesis of ((5-(pyridin-1-ium-1-ylmethyl)-1,3-phenylene)bis(methylene))bis(ethyl(methyl)sulfonium) (7b)

To a stirring solution of 1-(3,5-bis(bromomethyl)benzyl)pyridin-1-ium (3, 0.28 mmol) in CH₃CN/H₂O (3 : 1 in volume) was slowly added (dropwise) a previously stirring solution of ethanethiol (0.60 mmol) and sodium bicarbonate (0.60 mmol) in CH₃CN/H₂O (3 : 1 in volume) at room temperature. The resulting reaction mixture was stirred for 36 hours. After that, the solvent was removed under reduced pressure. Then, the reaction mixture was diluted with cold water and ethyl acetate. The organic layer was extracted with EtOAc (3 × 50 mL) and washed with brine, and dried over anhydrous Na₂SO₄. After that, the solvent was removed under reduced pressure to afford a white solid, which was used for the next reaction without further purification. Then, to the stirring solution of the above white solid 1-(3,5-bis((ethylthio)methyl)benzyl)pyridin-1-ium (0.06 mmol) (5b) in dry DCM was added AgBF₄ (0.06 mmol) and methyl iodide (0.16 mmol), and the resulting mixture was stirred for 5 hours at room temperature. TLC monitored the progress of the reaction. After total consumption of the starting compound, the solvent was removed under reduced pressure, and the resulting mixture was filtered off using a pad of celite to remove silver salt. Then, the crude mixture was washed with ether and CH₃CN to afford compound 7b as a brown gummy liquid. Characterization of the compound: dark brown gummy liquid (yield – 71%) ¹H NMR (600 MHz, DMSO-d₆) δ_ppm 9.15–9.08 (m, 2H), 8.69–8.61 (m, 1H), 8.23–8.21 (m, 2H), 7.95 (s, 1H), 7.58 (s, 2H), 5.93 (s, 2H), 4.71–4.62 (m, 4H), 2.89 (m, 2H), 2.77 (s, 6H), 2.73 (s, 2H), 1.33 (t, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ_ppm 162.8, 146.8, 145.5, 136.6, 133.8, 132.2, 130.9, 128.9, 66.1, 62.9, 59.3, 43.4, 35.5, 31.2, 21.2, 11.25, 8.84; ES-MS (ESI⁺) m/z: [(M + 3BF₄⁻] 889.525.

Synthesis of ((5-((4-(sulfomethyl)-1H-1,2,3-triazol-1-yl)methyl)-1,3-phenylene)bis(methylene))bis(dodecyl- (methyl)sulfonium) (8)

To a stirring solution of ((5-(azidomethyl)-1,3-phenylene)bis(methylene))bis-(dodecylsulfane) (4, 0.09 mmol) in DMF (3 mL) was added prop-2-yne-1-sulfonic acid (0.09 mmol) and the mixture was stirred for 10 minutes. After that, sodium ascorbate (0.003 mmol) and CuI (0.002 mmol) were added to the reaction mixture, and the solution was allowed to stir for 24 hours at room temperature. The reaction was monitored by TLC. After that, the unused solvent was removed under reduced pressure and diluted with ethyl acetate. The organic layer was separated, washed with brine, and dried over anhydrous Na₂SO₄. The solid crude product was washed with distilled hexane, the desired product 6 was almost pure, and the reaction mixture was used for the next reaction without further purification. To a stirring solution of compound 6 (0.58 mmol) in dry DCM, AgBF₄ (0.058 mmol) was added, followed by methyl iodide (0.15 mmol). The resulting mixture was stirred for 5 hours at room temperature. TLC monitored the progress of the reaction. After maximum consumption of compound 6, the solvent was removed under reduced pressure, and the resulting mixture was filtered off using a pad of celite to remove silver salt. Then, the crude mixture was washed with ether and CH₃CN to afford compound 8 as a brown gummy solid. Characterization of the compound: dark brown gummy solid (yield – 68%) - ¹H NMR (600 MHz, CDCl₃) δ_ppm 9.82 (s, 1H), 8.02 (s, 2H), 7.95 (s, 1H), 4.71–4.62 (m, 2H), 3.78–3.76 (m, 2H), 3.34–3.17 (m, 4H), 2.81 (s, 6H), 2.35–2.28 (m, 1H), 1.87–1.78 (m, 4H), 1.44–1.40 (m, 4H), 1.29–1.23 (m, 41H), 0.86 (t, 6H); ¹³C NMR (151 MHz, CDCl₃) δ_ppm 138.3, 133.0, 130.1, 129.0, 128.0, 125.3, 68.1, 47.2, 44.6, 41.4, 31.9, 29.7, 29.7, 29.6, 29.4, 29.0, 28.4, 25.6, 24.1, 22.7, 21.3, 14.1; ES-MS (ESI+) m/z: [(M/2 + 2BF4⁻ + Na⁺] 552.0522.

II. Zeta-potential and DLS measurement

The surface potential and hydrodynamic diameter of the compounds in the aqueous environment was measured by zeta potential and dynamic light scattering (DLS) using a Zeta sizer ZS90 (Malvern, Westborough, MA) instrument at 25 °C. The stock solution of the compound was prepared in chloroform, and multi-layered vesicles were prepared by rotary evaporation method with the help of a vacuum pump. To this dried compound, phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄ at pH 7.4) buffer was added and kept for hydration overnight at 50 °C. To obtain the unilamellar vesicles, a sample vial underwent vortexing and intermittent sonication. Disposable zeta cells (DTS1061) and a 3 mL fluorescence cuvette were used for the surface potential and DLS measurement.

III. Transmission electron microscopic measurements

The transmission electron microscope (TEM) was used to investigate the morphology of the aqueous soluble aggregates of the lipids. The lipid solution was prepared by the method mentioned above in PBS, pH 7.4. The freshly prepared lipid solution (without extrusion) was diluted to half of its original concentration using 1 mL PBS, pH 7.4. Then, 10 μL of lipid solution was taken and placed onto a carbon-coated copper grid and allowed to absorb for 1 minute. The grid was then carefully blotted with filter paper, and only a trace amount of the solution in the middle of the grid was kept. After that, the grid was allowed to dry for 10 minutes at 30 °C. Finally, 5–10 μL of 2% 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 were collected using a JEOL JEM 2100 transmission electron microscope (operated at a maximum accelerating voltage of 200 kV).

IV. Antimicrobial activity and bactericidal activity

The antimicrobial activities of the compounds were tested by using a broth dilution assay in 96-well plates. E. coli MTCC 1687 and P. aeruginosa MTCC 2488 were cultured in Luria Bertani (LB), and S. aureus MTCC 96 and methicillin-resistant S. aureus (MRSA) cells were cultured in Brain Heart Infusion (BHI) broth media until the mid-logarithmic phase at 37 °C in a shaker incubator and harvested by centrifugation. Then, the cells were washed with PBS buffer and suspended in the same buffer. In the presence of varying concentrations of the compounds, bacterial cells were incubated at 10⁶ CFU mL⁻¹ at 37 °C. After 14–16 hours of incubation, the optical density (OD) was measured at 600 nm by using a Tecan infinite M200 Plate reader. For the bactericidal activity test, the solution with no visible growth was transferred into a fresh well containing only media. The plate was incubated for 24 hours at 37 °C, and then the OD was measured. To get the closest minimum inhibitory concentration (MIC) value, this antimicrobial experiment was repeated at a variable concentration range, and was repeated thrice at the reported MIC value procedure.

V. Antibiotic loading and release profile study

The stock solutions of tetracycline and amoxicillin antibiotics were prepared in PBS. The compound was dissolved in chloroform and dried by the rotary evaporation method to form a multilayer film. To this dried vial containing a dried layer of the compound, the aqueous solution of antibiotic was added and incubated at 37 °C in a water bath for hydration. The liposomes underwent 7–8 freeze–thaw cycles, followed by vigorous vortexing for 2 hours for superior encapsulation of the antibiotics. The unloaded antibiotic was removed by centrifugation. The antibiotics loading efficacy was measured by a UV-vis spectrophotometer. The encapsulated liposome was treated with 5% Triton X-100 to break the liposome, and the released antibiotic was monitored by its absorbance. The absorption of the antibiotic alone was also recorded. The concentration of the loaded antibiotic was calculated by Lambert–Beer's law, A = ε·C_ab·l, where C_ab is the released antibiotic concentration from the liposome. For this calculation, the calibration curve was obtained to calculate the extinction coefficient (ε) by the absorption of the antibiotics at different concentrations. The drug release profile was observed at different time intervals up to 60 hours by fluorescence spectrofluorometer (for tetracycline, the λ_ex and λ_em values were 390 and 550 nm, respectively; for amoxicillin, the λ_ex and λ_em values were 390 and 550 nm, respectively³⁶). All of the measurements were performed in PBS. For 100% release of the antibiotic, the liposome suspension was treated with Triton X-100, followed by sonication and fluorescence measurement. The same experimental protocol measured the antimicrobial activity of the antibiotic encapsulated liposome.

VI. Antibiofilm activity study

The antibiofilm activity study was performed by the crystal violet staining method. S. aureus MTCC 96 is a well-known non-motile, sessile strain and forms a biofilm on the bottom of the wells. The bacterial cells were grown and treated with compounds, as mentioned in an earlier section. After the overnight incubation, the planktonic cells were pipetted out, and the wells were rinsed with PBS. The wells were air-dried under laminar airflow. Then, the crystal violet solution (1%, v/v) was added to each well, and the plate was incubated for 20 minutes. After incubation, the crystal violet solution was taken out, and the wells were dried. Then, ethanol (95%) was added to each well, and the absorbance was taken at 590 nm using a 96 micro-titer plate reader (Infinite M200, TECAN, Switzerland).

VII. Propidium iodide uptake assay

The stock solution (1.5 mM) of propidium iodide (PI) was prepared in sterile Milli-Q water and stored at 4 °C. The S. aureus MTCC 96 cells were cultured overnight and collected by centrifugation, and resuspended in the same media. Cells were again diluted in media at 10⁶ CFU mL⁻¹. After the addition of the compound, the cells were incubated at 37 °C at 180 rpm. The sample (1 mL) was collected at the 1st, 2nd, and 3rd hour. The culture was centrifuged and washed with PBS, and the cells were incubated with PI at 30 μM concentration for half an hour. Cells were again centrifuged, washed, and resuspended with PBS, and the fluorescence measurement was recorded at an excitation wavelength of 535 and emission wavelength of 617 in a spectrofluorometer (HORIBA, flioroMax-4).

VIII. Fluorescence imaging assay

The PI and cFDA-SE dyes were used to stain the dying and viable bacterial cells. The S. aureus cells were cultured and harvested, as mentioned in an earlier section. Cells were treated with the compound, and only buffer was added to the cell culture as a control. After 5 h of incubation, the cells were collected by centrifugation and washed with buffer, and diluted up to 10⁶ CFU mL⁻¹. The cells were separately stained with PI at a concentration of 30 μM and cFDA-SE at a concentration of 10 μM. After 30 min of incubation, 10 μL of the stained sample was taken on a thin glass slide and observed under the fluorescence microscope.

IX. Morphological study

The morphological assessment was done by field emission scanning electron microscope (FESEM). The S. aureus MTCC 96 and E. coli MTCC 1687 cells were cultured and harvested by centrifugation at 5000 rpm for 5 minutes. Cells were washed and treated with the compound, and the control was taken as the bacterial culture without compound treatment. After 5 hours of incubation, cells were collected by centrifugation, washed with Milli-Q water, drop-casted on an aluminum foil studded glass grid, and air-dried under laminar airflow. The drop-casted sample was again mounted on the FESEM metal grid sandwiched by carbon tape before the FESEM analysis sample was double-coated by gold.

X. Membrane depolarization study

For this study, S. aureus and MTCC 96 cells were grown as mentioned in the above section until the mid-log phase and harvested, followed by resuspension in HEPES buffer (10 mM HEPES and 50 mM glucose). To this, DiSC₃5 was added at a final concentration of 0.4 μM, and cells were incubated for 1 h. After this, KCl was added to the cell suspension at a concentration of 100 mM. After 10 min incubation, variable concentrations (0, 2, and 4 μM) of the compound and 30 μM valinomycin as a positive control were added to the cell suspension, and the fluorescence spectra were recorded at excitation and emission wavelengths of 620 nm and 650 nm, respectively.

XI. Cytocompatibility test

To test the viability of human cells in the presence of the antimicrobial drugs, HeLa cells (1 × 10⁴ cells/well) were seeded into 96-well plates in 100 μL of DMEM per well and left overnight for attachment. The cells were then treated with variable concentrations of the compound, and only buffer as the control. After incubation for 24 h, 10 μL MTT reagent (5 mg mL⁻¹) was added to each well (containing cells in DMEM media with compounds), and then incubated for another 3 h at 37 °C in a CO₂ incubator. Later, the MTT reagent was removed from the wells, and formazan crystals were dissolved by adding 100 μL of dimethyl sulfoxide (DMSO) in each well. The absorbance was recorded at 570 nm to measure the cell viability by using a plate reader.

XII. Haemolytic assay

To test the toxicity of the compound against the blood cells, haemolysis screening was performed according to the reported protocol. The haemolytic assay was performed with erythrocytes extracted from fresh human blood. Blood was centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded, and cells were washed with PBS. The 5% haematocrit was prepared in the same media. Serially diluted sulfonium compounds were added in a vial up to 240 μL, including a negative control with only buffer and positive control with 0.5% Triton X-100 and 60 μL of the 5% haematocrit was added to each vial, and incubated for 1 hour at 37 °C. After incubation, the vials were centrifuged at 1500 rpm for 10 minutes, and 50 μL of the supernatant was added to fresh 96-well plates. The absorbance was recorded at 410 nm. The% haemolysis was calculated by comparing the absorbance of the sample with the positive control and the absorbance of the negative control. All experiments were performed in accordance with the Guidelines of the Institutional Human Ethics Committee (IHEC), and the experiments were approved by the ethics committee at Indian Institute of Technology Guwahati. Informed consents were obtained from the human participants of this study.

Conclusions

Bacterial infections and antibiotic resistance are rapidly becoming one of the most daunting healthcare burdens of our day, which immediately need effective therapeutic strategies. The current study demonstrates that the modular synthesis of membrane-active sulfonium-based lipids can effectively form liposomes, and encapsulate the commercially available antibiotics to have a synergistic effect against bacterial strains responsible for respiratory infections like cystic fibrosis. Liposomal aggregates are the rising armour to strive against such respiratory syndromes. The fascinating properties of the antibiotic-encapsulated liposomes were the prudent assimilation of two weapons, which permitted harnessing their concomitant activity. The membrane-directed properties of the lipids make them less prone to drug resistance, and the most potent lipid also augments the activity of commercial antibiotics. We envisage that this inherent antimicrobial drug carrier can be proven as the panacea towards the path of antibiotic stewardship.

Conflicts of interest

There are no conflicts to declare.