Insights into the Formations of Host–Guest Complexes Based on the Benzimidazolium Based Ionic Liquids−β-Cyclodextrin Systems

Abstract

Inclusion complexation is one of the best strategies for developing a controlled release of a toxic drug without unexpected side effects from the very beginning of the administration to the target site. In this study, three benzimidazolium based ionic liquids (ILs) having bromide anion and cation bearing long alkyl chains, hexyl- ([C₆CFBim]Br), octyl- ([C₈CFBim]Br), and decyl- ([C₁₀CFBim]Br) were designed and synthesized as antibacterial drugs. Inclusion complexes (ICs) of studied ILs have been prepared by the combination of β-cyclodextrin (β-CD), considering these conjugations should enhance the benignity of ILs and make them potential candidates for the controlled drug release. Characterizations and structural analysis of studied ICs have been performed by ¹H NMR, 2D-ROESY NMR, FT-IR, HRMS, TGA, DSC, surface tension, ionic conductivity, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC). Further, the morphology of the ICs has been analyzed by SEM and TEM. Furthermore, neat ILs and ICs have been treated against Escherichia coli and Bacillus subtilis to investigate their antibacterial activity, which confirms the prevention of bacterium growth and the shrinkage of the bacterial cell wall. The findings of this work provide the proof of concept that studied benzimidazolium based ILs-β-CD host–guest complexes should act as a potential candidate in controlled drug delivery and other biomedical applications.

1. Introduction

A continuous search for novel classes of antibacterial agents is essential as pathogenic bacteria become resistant to most antibiotics. Compared to conventional antibacterial agents, novel materials should possess long-lasting activity, significant efficiency, high biocompatibility, and facile synthetic strategy. Recently, a few researchers explored a novel class of organic salts, ionic liquids (ILs), as potential candidates as antibacterial agents, mainly due to their tunable physical, chemical, and biological properties by altering either the structure or the type of cation and anion.¹ Among various classes of ILs, imidazolium-based ILs have gained substantial interest for applications in pharmaceutical and related research areas.¹⁻⁴

Further, imidazolium-based ILs were explored in silico and in vitro studies,⁵ bioactive substances for combating nosocomial infections,⁶ and antiproliferative agents against T98G Brain Cancer Cells.⁷

Recent findings have suggested that substituted imidazole derivatives, mainly benzimidazolium ILs can act as highly effective antibacterial agents because of having a cationic headgroup and hydrophobic alkyl chain.⁸⁻¹⁰ Further, disubstituted benzimidazolium ILs are potential candidates for their extended applications in the field of antibiofilm and antibacterial implementation.⁸ Additionally, benzimidazolium salts exhibit in anticancerous and antiviral actions.¹¹ Lu et al. have demonstrated anticancerous activity of carbazole substituted benzimidazolium for HL-60, MCF-7, SMCC-7721, and SW480 cell lines.¹²

Generally, most drugs have undesirable side effects as they interact with other body parts. These side effects hinder developing a strategy for optimum medications for several diseases, for example, infectious diseases, neurodegenerative diseases, and cancer.¹³⁻¹⁵ To overcome this aspect, significant efforts have been focused on preparing inclusion complexes (IC)¹⁶⁻¹⁹ by combining the drug and cyclodextrins (CD) as a safe drug delivery pathway. Cyclodextrin-based inclusion complexes can camouflage the unexpected characteristics of the drugs and generally cause a synergistic effect. Also, cyclodextrin-based derivatives could be used as antibacterial drug carriers as they are capable of identifying specific cellular receptors because of the presence of lectins at the surface of a bacterial cell.^20,21 Thus, this strategy may find further developments in the delivery of toxic antibacterial drugs without undesirable side effects from the beginning of the administration to the target site. Zhou et al. have demonstrated that skin irritation can be reduced by IC formation with CD when long alkyl-chain oligomeric ammonium salts show potent antimicrobial activity.²² Hodyna et al. have examined 1-dodecyl-3-methylimidazolium tetrafluoroborate IL which exhibited highly efficient antimicrobial activity and reduced toxicity for IC with β-cyclodextrin.²³ Gravel et al. have shown a controlled drug delivery system based on an antibacterial imidazolium salt as a drug, β-cyclodextrin, and a competitive nontoxic guest.²⁴

However, until today, only limited reports are available regarding the detailed investigation, applications of ICs formation by β-CD with benzimidazolium ILs, leaving sufficient room for exploring the structure–property relations of IC between ionic liquid and β-cyclodextrin.

In the present study, we have synthesized three benzimidazolium ILs, hypothesizing that these ILs having a long alkyl chain with a cationic headgroup, which is expected to show substantial antibacterial activity. In addition, to understand the structural-activity relation (SAR), the alkyl chain length has been varied from hexyl to decyl. Further, for making the ILs less toxic, we have prepared the inclusion complex with β-CD.

Interaction between β-CD and benzimidazole-based ILs has been examined by ¹H-NMR, HRMS, and IR spectroscopic techniques. Thermodynamics insights into the interactions and the stoichiometry of the inclusion of IL-CD complex have been performed by isothermal titration calorimetry (ITC) study. The effects of surface tension and conductance have been investigated by surface tension and conductivity experiments. The thermal stability of the ICs has also been characterized by TGA and DSC. Further, their morphology has been characterized by SEM and TEM. The effect of the size on the addition of β-CD has been examined by DLS. The antibacterial activities of the ILs and the effect upon complex formation by the IL-CD against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) have been examined by colony-forming units (CFUs) and scanning electron microscopy (SEM). The antibacterial activity of the ILs and its β-CD conjugates has also been examined by the zone of the inhibition technique.

To the best of our knowledge, this is the first report which introduces a drug delivery system incorporating benzimidazolium IL into the β-CD based host where ILs acting as model drugs show antibacterial properties. Hence, this work illuminates a new light on the immense possibilities of progressive drug delivery and biomedical applications using ionic liquid as a drug with rational structural modifications.²⁵⁻²⁷

2. Experimental Section

2.1. Materials

β-CD was obtained from Sigma-Aldrich (99% pure). 3-(2-Carboxyethyl)-1-decyl-1H-benzo[d]imidazol-3-ium bromide and 3-(2-carboxy ethyl)-1-hexyl-1H-benzo[d]imidazol-3-ium bromide were obtained from Sigma-Aldrich. The chemicals used for synthesizing the given ILs are benzimidazole (Lobachemie; 99%), 1-bromohexane (Spectrochem; 98%), 1-bromooctane (Spectrochem; 98%), 1-bromodecane (Spectrochem; 98%), and 3-bromopropionic acid (Alfa Aesar; 97%).

2.2. General Experimental Methods

The ¹H NMR has been recorded in a Bruker Avance 500 MHz spectrometer with DMSO-d₆ as an external solvent. The ¹H and 2D-ROESY NMR of the ICs have been acquired by using a high-performance digital NMR spectrometer (Bruker Avance 500). All the measurements have been carried out at room temperature with D₂O as the NMR solvent. IR spectra have been recorded using a JASCO FT-IR-4100 (Fourier-transform infrared) spectrometer in the range of (4000–400 cm^–1) with a resolution of 4.0 cm^–1. Electrospray ionization mass spectra (ESI-MS) have been recorded on a micromass Q-TOF LC/MS. Specific conductance of the solutions has been measured by “Mettler Toledo” Seven Multi conductivity meter having uncertainty (1.0 μS m^–1). HPLC grade water having specific conductance of 6.0 μS m^–1 has been used throughout the whole experiments. Calibration of the conductivity cell was performed via a 0.01 M aqueous KCl solution. Surface tension study has been recorded at the spinning drop video tensiometer (SVT 20 N, Dataphysics, Germany). The tensiometer was calibrated before each measurement.

ITC study has been carried out taking a freshly prepared aqueous solution of IL in a MicroCal iTC200 Malvern instrument. All the ILs solutions were degassed before the experiment. The sample cell was loaded with 1.5 mM, and the aqueous solution of β-CD (conc. 15 mM, 2 μL titrant volume for each injection) was injected using an injection syringe with continuous stirring at 700 rpm. Both the titration experiments were performed at 25 °C. All the data were evaluated by MicroCal analysis software and fitted as illustrated in other reports.²² P-XRD patterns of the compounds have been recorded by using a Bruker D8 Advance X-ray diffractometer using Cu Kα radiation (λ = 1.54178 Å). The morphologies of the ILs and ICs have been analyzed using FEI-Quanta FEG 200F scanning electron microscopy (SEM), and high resolution-TEM (HR-TEM) images of the samples have been obtained using a JEOL 3010 instrument with a UHR pole piece. TGA has done by TA Instruments Hi-Res. (TGA Q500). Samples were heated at 20 °C/min from 25 to 600 °C in a dynamic nitrogen atmosphere.

A DSC instrument (TA Instruments DSC Q200) has been used to determine the Phase transition having sensitivity 0.2 μW and temperature accuracy ±0.1 °C.

For the determination of the zone of inhibition, bacteria were separately cultured in LB media and log-phase bacteria were spread on Mueller-Hinton agar (MHA) plates using a sterile L spreader. Then the drugs were soaked on filter paper and placed on the bacteria-spread MHA plates. The plates were then incubated at 37 °C. After 16 h the zone of inhibition (if any) was measured.

Additionally, for the determination of the minimum inhibitory concentration (MIC⁹⁹, the minimum concentration at which 99% bacteria were killed) bacteria were separately cultured in LB media, and log-phase bacteria were taken to inoculate (2% inoculums) the sterile LA media in which drugs of the different concentrations were also mixed. After 16 h, of incubation, the broths were checked for any visible growth. For assessing the living bacteria, the media was serially diluted and spread on Luria agar (LA) plates for the determination of colony-forming units (cfu).

Further, for investigating the impact of drugs (ILs/ICs) on bacteria, SEM images were collected by using JSM-6360. Briefly, 3% glutaraldehyde and 1% osmimun tetraoxide have been used for fixation of the bacteria and then dehydrated the samples using an ascending alcoholic gradient. The samples of bacteria which were mounted on glass slides were sputter-coated with gold for 1 min and then loaded on a metal stab with a carbon tape for SEM.

2.3. Synthesis of Benzimidazole-Based Ionic Liquids

Carboxyl-functionalized benzimidazolium ionic liquids (CFBILs) have been synthesized in two steps. Initially, in the first step, benzimidazole was treated with 1-bromoalkane followed by a reaction with 3-bromopropionic acid.

2.4. Preparation of Alkyl Benzimidazole

The alkyl benzimidazole was synthesized by an alkylation reaction of benzimidazole. In a 250 mL double-necked round-bottomed flask benzimidazole (50 mmol) and potassium hydroxide (100 mmol) were added in acetonitrile (70 mL) and were stirred for 2 h at 80 °C. To this reaction mixture 1-bromoalkane (51 mmol) was added dropwise for 30 min and then stirred for 48 h, under N₂ atmosphere. Then the solvent was evaporated using a rotary evaporator when the reaction mixture turned into room temperature to get a syrupy liquid. Then after the DCM–water workup, the organic layer was collected and dehydrated by Na₂SO₄. The remaining solvent was removed to get the crude product. The unreacted starting material and moisture were removed by applying a high vacuum at 70 °C for 48 h (Scheme 1).²⁸

Scheme 1. Reaction Scheme for the Synthesis of Alkyl Benzimidazole.

2.5. Synthesis of CFBILs

The preparation of CFBILs (carboxy functionalized benzimidazolium ILs) was carried out by adding toluene (80 mL) to the alkyl benzimidazole (51 mmol), which was stirred for 1 h at 70 °C. 1-Bromopropionic acid (50 mmol) was added to it dropwise for about 30 min. Subsequently, the solution was kept for stirring for another 48 h at 60 °C. The product obtained was separated by the decantation of toluene directly from the reaction flask. Then it was washed three times first by using ethyl acetate (50 mL) followed by diethyl ether (50 mL). Finally, a high vacuum was applied for about 24 h at 60 °C. The hexyl derivative was obtained as a light brown solid while the remaining octyl and decyl derivatives were highly viscous, gel-like products. Dried ILs were then characterized using ¹H NMR (Figure 1 and Figure S1(a), S2(a) and S3(a)), ¹³C NMR (Figure S1(b), S2(b), and S3(b)), HRMS (Figure S4) and IR (Table S1), which confirmed the absence of residual benzimidazole or acid (Scheme 2).²⁹ The purity of the synthesized ILs was found to be ≥97%.

Figure 1.

Proton NMR spectra of free β-CD, [C₁₀CFBim]Br, and B10 in D₂O.

Scheme 2. Synthetic Scheme of CFBILs.

2.6. Synthesis of ICs

Each IC was prepared by the kneading method.³⁰ Equimolar quantities of IL and β-CD were mixed. A homogeneous paste was prepared in a mortar by adding small amounts of water with continuous kneading for half an hour.

Each IC is named B6, B8, and B10, where B refers to β-CD, and the respective integers denote the number of the carbon atom of the alkyl chain present in the benzimidazolium ionic liquids (Table 1). As synthesized, three ILs showed three different phases of their existence (i.e., solid, semiliquid, and gel-like material was found for ICs) (Figure 1). In the case of B6 and B8, IC phases were precipitated after 2 days from synthesis, and different phases come out after the next two consecutive days. The IC phases were obtained by evaporating the aqueous solution in a rotary evaporator and, subsequently, a high vacuum at 80 °C. Although the IC phases were translucent in solution, finally they turned into dark-colored (i.e., deep brown, straw yellow, and metallic golden) solid (B6 and B8) and gel (B10) (Figure S5). Finally, ICs were characterized using ¹H NMR (Figure 1, Figure S6(a) and Figure S6(b)), 2D-ROESY (Figure 2, Figure S7(a), and Figure S7(b)), IR (Figure 3 and Table S1), and HRMS (Figure S8(a), S8(b), and S8(c)).

Table 1. Nomenclature of Inclusion Complexes and Corresponding ILs.

Name of the CFBILs	Corresponding IC	Name of the IC
[C6CFBim]Br	[C6CFBim]Br-β-CD	B6
[C8CFBim]Br	[C8CFBim]Br-β-CD	B8
[C10CFBim]Br	[C10CFBim]Br-β-CD	B10

Figure 2.

2D ROESY spectrum of B10 in D₂O.

Figure 3.

Stacked FT-IR spectra of β-CD (bottom), [C_nCFBim]Br, and Bn where (n = 6, 8, 10 bottom to top, respectively).

3. Results and Discussion

3.1. Inclusion Complex Formation by ¹H NMR Analysis

To understand the IC formation, a stack plot of solution-state ¹H NMR spectra for β-CD, [C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br has been examined (Figure S6(a), Figure S6(b), and Figure 1, respectively). It is well-known that β-CD assumes the torus conformation where both H-3 and H-5 protons are situated within the cavity and H-2 and H-4 protons are positioned at the outer surface of the torus. The overlap of host protons in ICs with H-5 and H-6 signals of free β-CD undoubtedly shows that all resonances and signals appear as a mixture of multiple as well as broad peaks. After the addition of ILs to the solution of β-CD, the H-5 shift stimulates to upfield. Comparative results indicate a significant upfield shift for the intracavity H-3 and H-5 protons of β-CD in ICs rather than that of its free form implies the noticeable changes in the microenvironment of IL, which validates the inclusion of the ILs into CD. Protons of the primary alcoholic functional group H-6 appear close to the narrower rim, and hence, the upfield shift is not as evident as H-5 of β-CD. H-2, H-4 protons of β-CD are positioned in the outer surface of the rim, and signals do not change even after the addition of IL. Results specify that the guests are incorporated into the cavity of β-CD. Furthermore, the signals of the three ILs have altered in β-CD, and significant alkyl peaks of [C₁₀CFBim]Br are shifted to a large extent as shown in Table 2. No signal changes were noticed for a proton of the benzimidazolium ring, indicating that this part of the IL is free. Hence, it can be inferred that exclusively the long-alkyl chain of [C₁₀CFBim]Br was included in the cavity of β-CD.³¹ It can be concluded that the longer alkyl chain plays a key role in the formation of the strong inclusion complex.

Table 2. Variation in Chemical Shifts (ppm) between Pure β-CD Molecules and Three Different ICs in D₂O.

Protons of β-CD	B6	B8	B10
H-3	0.05	0.13	0.14
H-5	0.02	0.04	0.07

3.2. 2D-ROESY Spectra Analysis

ROESY spectrum analysis is valuable for determining proton signals that are in the same environment and close to one another, even though they remain nonbonded. So, this type of spectrum can confirm the formation of the inclusion construction of ICs. 2D-ROESY spectra for B6, B8, and B10 are given in Figure S7(a), Figure S7(b), and Figure 2, respectively. Results are similar to proton NMR; correlations for H-3, H-5 protons denote the interactions among H-3, H-5 of β-CD with −CH₂ groups in the alkyl series of guests, i.e., ILs. The results predict that the alkyl chain of each IL was present in the interior of the hydrophobic cavity, and the benzimidazolium ring projects toward the outer face. Consequently, opposite orientations for ILs to form ICs (1:1) inclusion ratios are possible.³¹

3.3. FT-IR Analysis

Infrared spectroscopy helps to describe the position of the functional group and moreover indicates the possible functional group interactions. Inclusion complexes between the ILs (Figure 3 and Table S1) and β-CD can also be determined and characterized by this technique. For the host or guest, IR peaks with changes in shape, intensity, and shift can be used to detect the formation of an inclusion complex.

Figure 3 shows β-CD absorption peaks for C–H, C–O, and C–O–C at 2921.12 cm^–1, 1153.45 cm^–1, and 1021.32 cm^–1, respectively, which are shifted to 2927.89 cm^–1, 1158.75 cm^–1, and 1076.93 cm^–1 in B6. Moreover, the sharp and narrow infrared bands denote the weak intermolecular interactions with β-CD.

Also, the plot below displays that the absorption peak of O–H in β-CD at 3349.23 cm^–1 has been shifted to 3418.06 cm^–1 in B8. The peak at 2920.12 cm^–1, which is the stretching vibration of −C–H from −CH₂ of β-CD, is shifted to 2926.69 cm^–1. C–H in-plane bending of pure IL appears at 1135.22 cm^–1, which is shifted to 1268.18 cm^–1. The peak at 735.85 cm^–1, which is due to the C–H out of plane bending in the complex, is shifted to 766 cm^–1. The results show that the interactions in the inclusion complex are stronger compared to those in B6.

In the case of B10 complex, pure β-CD shows a peak at 3349.23 cm^–1 for the O–H vibration, which is shifted to 3244.18 cm^–1. Additionally, the peak at 1732.14 cm^–1, which is the stretching of C–H for the pure IL, is shifted to 1623.98 cm^–1. The C–O–C stretching vibration peak at 1024 cm^–1 shifts to 1031.32 cm^–1 in the complex. The peak at 754 cm^–1 (C–H out-of-plane bending) is changed to 751 cm^–1 in the complex.

The broad-band obtained at the 3400–3300 cm^–1 region may be due to the formation of extended noncovalent interactions among the carbonic group of the carboxyl group attached with the quarternized N atom of the heterocycle and O–H groups of the outer rim of β-CD (Scheme 3).³²

Scheme 3. Extended Noncovalent Interactions between the Carboxyl Group at the Cationic N Center of the Imidazole Ring and the O–H Group (Outer Rim) of β-CD.

Additionally, the fact that there was no change in the IR peaks of the benzimidazole ring proves that the benzimidazolium part of ILs remains outside of the CD cavity. Moreover, a large shift of the alkyl −C–H stretching vibrational band frequency for −CH₃ and −CH₂ of the alkyl part in the case of B8 and B10 over the pure ILs indicates that the alkyl chains in ILs move in the inner cavity by van der Waals (vdW) noncovalent hydrophobic–hydrophobic interaction, in the hydrophobic interior of cyclodextrin.³³

Further, among all the three ILs, the maximum broadening obtained at the 3400–3300 cm^–1 region for B10 indicated that [C₁₀CFBim]Br was strongly associated with the β-CD molecule to be considered as the firm IC.³⁴⁻³⁶

3.4. ESI-MS Study

The positive mode “ESI-MS” study has enormous importance to inspect the host–guest complexation with the three studied ILs. Mass spectrometric analyses have been used to confirm the formation of ICs (Figure S6(a), S6(b), and S6(c)). Observed peaks verify that in the case of B6, B8, and B10, the desired ICs have been formed in a (1:1) (host:guest) stoichiometric ratio. Mass spectra of (1:1) stoichiometries of [β-CD:{[C₆CFBim]Br}], [β-CD:{[C₈CFBim]Br}], and [β-CD:{[C₁₀CFBim]Br}] systems were evaluated by ESI-MS which denotes all the favored expected masses (Table 3). These results taken together recommended that the cationic part of ILs concurrently inserted in the β-CD hollow space with a (1:1) stoichiometric ratio.³¹

Table 3. Experimental m/z Values for Different Ions of the ICs.

IC	Ions	m/z
B6	[[C6CFBim]+ + β-CD+H+]2+	1409.54
	[[C6CFBim]+ + β-CD+6H+]7+	1438.07
B8	[[C8CFBim]+ + β-CD+H+]2+	1437.57
	[[C8CFBim]+ + β-CD+5H+]6+	1443.67
B10	[[C10CFBim]+ + β-CD+H+]2+	1465.60
	[[C10CFBim]+ + β-CD+16H+] 17+	1481.78
	[[C10CFBim]+ + β-CD+18H+] 19+	1483.78

3.5. Conductivity

Conductivity measurement is a vital tool for investigating the host–guest-based inclusion phenomenon. This also can provide information about the stoichiometry of the complex formation. Arrangements between β-CD and ILs have a significant effect on the conductivity of the solutions. Conductivity experiments have been performed for three selected ILs [C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br with the increased concentration of β-CD in water at 298.15 K shown in Figure 4(a), (b), and (c).

Figure 4.

Plots of specific conductance (κ) versus concentration of β-CD at different concentrations of (a) [C₆CFBim]Br and (b) [C₈CFBim]Br and (c) [C₁₀CFBim]Br at 298.15 K.

Inspection notifies that each plot displays a break point in their conductance at a definite concentration. Elaborately, the conductance decreases with increasing concentration and then attains a particular point (breaking point) and formerly lowers down, which indicates the construction of an inclusion complex (Tables S2, S3, and S4). This can be ascribed as, with the increased addition of CD, the IL dispersed in the aqueous medium having a long hydrophobic tail enters into the hydrophobic cavity of CD and shows a sudden decrease in their conductance value at this certain concentration. The decrement of conductance can be described as the constrained motion of the IL when it enters into the cavity which eventually decreases the free IL concentration, responsible for the conductivity of the medium.³⁷ Also, this sudden decrease in conductance generating a breaking point shows the indication of forming a 1:1 host–guest inclusion complex.³⁸

3.6. Surface Tension

Surface tension is also an important technique for investigating the inclusion complexation as well as for determining the stoichiometry between the host and guest. Here the ILs used for the present study are surface-active in aqueous solution. When the aqueous solution of ILs was treated with the increased concentrations of CD (Tables S5, S6, and S7), there was a sharp increase in the surface tension of the resultant solution mixture along with the generation of one breaking point in each case (Figure 5(a), (b), and (c)).³⁹⁻⁴²

Figure 5.

Graphs representing surface tension (γ) versus concentration (M) of β-CD at 298.15 K for (a) [C₆CFBim]Br, (b) [C₈CFBim]Br, and (c) [C₁₀CFBim]Br.

This can be attributed as the ILs dissolved in an aqueous medium because of having a long hydrophobic tail and cationic headgroup likely to develop themselves at the air–water interface for forming an adsorption monolayer. With the increased addition of CD, the surface-active ILs enters into the cavity of CD by breaking the monolayer. This leads to the formation of water-like surface tension, and consequently, the surface tension of the resultant solution increased with the increase of CD concentrations.⁴¹ Here also one breaking point in every case designates 1:1 inclusion complex formation.

3.7. ITC Measurements

ITC is such a well-grounded thermodynamic method by which a single experiment can endow a complete thermodynamic picture of a host–guest interaction. Thus, to further confirm the binding stoichiometric ratios and binding constants, ITC experiments have been performed at 25 °C between all three ILs (1.5 mM) and β-CD (15 mM) shown in Figure 6(a), (b), and (c). Only for [C₁₀CFBim]Br a typical S-shape titration isotherm curve has been obtained in Figure 6(c).

Figure 6.

ITC data for the titration of (a) [C₆CFBim]Br, (b) [C₈CFBim]Br, and (c) [C₁₀CFBim]Br with β-CD in water.

All three titration curves fitted well with the single-site binding model²² described in Appendix S1. Fitting parameters, viz. binding constant (K_b), no. of binding sites (N), enthalpy of binding (ΔH_b), and entropy of binding (ΔS_b), obtained are given in Table 4. K_b values for C₂mimTf₂N to C₈mimTf₂N⁴³ are in the range from 2740 to 9808 M^–1, and those for C₂mimPF₆ to C₆mimPF₆⁴⁴ are in the range from 170 to 401 M^–1. The bolaform covalent amphiphile bipyridinium bromide with β-CD⁴⁵ has shown a K_b value of 5910 M^–1, which is in good comparison (5800 M^–1) with the B10 IC studied in this work. These results indicate that K_b values, apart from complexing agent, are highly dependent on the nature of the cation and anion of the ILs.

Table 4. Fitted Parameters Obtained from the ITC Experiment for Different ICs.

IC	ΔHb(cal/mol)	ΔSb(cal/mol/deg)	TΔSb(cal/mol)	Kb(M–1)	N (Sites)
B6	–948 ± 28	6.56	164	134 ± 32	1.14 ± 0.24
B8	–1678 ± 35	7.89	197	901 ± 29	1.01 ± 0.01
B10	–2261 ± 36	9.64	241	5800 ± 38	1.03 ± 0.01

The results in the present study indicate that the IC formation is an enthalpy driven process.⁴⁶ Additionally, ΔH binding turned more negative when the long alkyl chain of the benzimidazolium ring was progressively extended, attaining the most exothermic association with the decyl derivative. Besides, electrostatic attraction of benzimidazolium cation and extended H-bonding between the carboxyl groups of ILs and −OH groups of the exterior part of the β-CD also imparts stability for the IC formations.

Thus, the ITC experiment supports the conductance, surface tension, and ESI-mass analysis and proves that all three ILs form 1:1 IC formation with β-CD. Besides, this experiment also trends with the chemical shift from ¹H NMR and shows that out of three ILs [C₁₀CFBim]Br forms from strong IC formation with β-CD.

3.8. P-XRD Study

P-XRD is a powerful technique for studying ICs to evaluate their structure and verify the formation of the ICs when compared with parent molecules. It is well-known that the crystal structures of β-CD-based ICs are primarily of three kinds: viz. channel, layer, and cage. P-XRD patterns of β-CD, surface-active ILs, and their particular ICs have been demonstrated by the stack plot of P-XRD in Figure 7. ICs of B6, B8, and B10 produce dissimilar patterns, designating a head-to-head channel-type structure, which is distinct from that of β-CD. This consequence is similar to the literature, which reveals that the ICs have an isomorphic and channel-like arrangement.⁴⁷

Figure 7.

P-XRD patterns of β-CD, B6, B8, and B10.

Generally, in the case of a guest molecule having a long alkyl chain, ICs form a head-to-head channel-type of the structure where β-CD molecules are assembled along an axis to develop a cylindrical structure. Water molecules arbitrate hydrogen bondings (H-bonding) among −OH groups of neighboring β-CD molecules. In assorted cases, H-bonding between β-CD imparts a crucial role in the stabilization of the complexes. van der Waals forces of interactions, the liberation of CD strain energy, and geometric compatibility are significant factors for the complexation. For the current explored three systems, hydrophobic interaction for surface-active ILs [C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br and β-CD can form channel-type structure.^{32,39,48−51}

3.9. SEM Analysis

Scanning electron microscopy study has been performed for verifying and differentiating surface features of β-CD-benzimidazole-based IL polymers with pure β-CD and pure ILs (i.e., [C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br) (Figure S9(a), (b), (c), and (d), respectively).

SEM images of all the ICs are shown in Figure 8(a), (c), and (e). Figure 8(a) shows layered cylindrical structures for B6. Figure 8(c) reveals the increment of the pore size of the β-CD based IC B8 in the presence of benzimidazolium ILs. In contrast, Figure 8(e) shows cluster type of structure in the case of B10 compared to pure ILs, and the literature supports this observation. Three pure ILs reveal attenuation to the crystal structure. They display the destruction of spherical nature, even surface, and smaller-sized particles, as shown in Figure S9(b), (c), and (d), while the three ICs in Figure 8(a), (c), and (e) exhibit a different crystalline structure and morphology. Distant morphologies were noticed for different ILs because of their distinctive characteristics.

Figure 8.

SEM and TEM images of B6 (a, b), B8 (c, d), and B10 (e, f), respectively.

The as-synthesized ILs having long alkyl chains form tape-like fibril aggregates through vdW interaction among the terminus alkyl chains which intertwist into a three-dimensional network forming highly microporous polymer-like material (where the diameter of the pore is >50 μM (Figure S9(d)). Additionally, hydrogen-bonding interactions persuade phase separation. The presence of β-CD, in polymer-rich benzimidazole-based ILs, shows highly ordered domains presumably owing to the rigidness of β-CD itself. Execution of the polymerization-based inclusion complexation and elimination of the solvent (H₂O) did not alter the micropores.⁵²⁻⁵⁴

3.10. TEM Analysis

TEM is a consistent process to study aggregation and complexation of β-CD and the ILs formed in the aqueous phase. The morphologies of the obtained ICs have been reflected in Figure 8(b), (d), and (f), respectively. In the case of B6, we have noticed both a fibrous as well as a cluster portion. Second, in B8 small spheres representing probably micelle characteristics have been found. Moreover, in B10, somewhat bigger spheres compared to B8 predicting the organization of micelles have been observed in Figure 8(f).

3.11. Thermogravimetric Analysis

TGA analysis has been performed for determining the thermal stabilities of three ICs, and results were evaluated with pure β-CD and pure [C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br. Figure S10(a), (b), and (c) illustrates the weight-loss curves of ICs. It is interesting to note that the initial decomposition temperature of all the ICs has a lower value than both pure ILs and β-CD. Generally, ICs show higher initial decomposition temperatures than those of β-CD, and the inclusion complexation is assumed to impart enhanced stability of β-CDs. Hence, these current TGA results are, to some extent, abnormal. This trend derives from the distinctive molecular structure and characteristics of benzimidazole-based ILs as compared to organic and polymer materials. This instability of the ICs is probably due to geometric distortion and steric congestion of the components. Furthermore, in all three cases, when only the alkyl chains are incorporated into the hydrophobic cavity of β-CD, both the cationic and anionic parts might be separated at a larger distance than a normal ion pair distance. This result suggests that hydrogen bonding acts as a secondary factor to sustain the development of ICs since a larger number of H-bonds would enhance the stability of the substrates. On the other hand, if some severe interactions, for example, hydrogen bonding, furnish the stability of inclusion complexes, the recovery would not be so easy when guest molecules are incorporated into the cavity of CDs. From this specialty, our present study shows the attention on the recovery and controlled release of these surface-active ILs.³⁹

3.12. Differential Scanning Calorimetry

It is noticed from Figure S11(a), (b), and (c) that the native ICs show polymer-like behavior and display different trends in the three different cases with B6, B8, and B10. The B10 IC shows more polymeric characteristics and involves three stages in the DSC analysis, while B6 and B8 show less polymeric properties. Various starting endothermic peaks had been observed at different temperatures that can be ascribed to the elimination of water, while the melting ranges of the samples have been assigned by the second endothermic domain in each curve.

It has been found from TGA that the stability of the ICs is less than ILs, especially in the case of B10 in comparison to native β-CD. This may be because of the characteristics of ILs which assist the nonvolatility and a higher temperature range of the ICs.⁵⁰

3.13. Dynamic Light Scattering

To investigate the IC formation as well as to determine the average hydrodynamic diameter of the IC formation, DLS studies were performed in water (Figure 9).

Figure 9.

Size distribution of (a) [C₆CFBim]Br, (b) [C₈CFBim]Br, and (c) [C₁₀CFBim]Br in the presence of increasing concentrations of β-CD.

According to the DLS results, it is clear that the average hydrodynamic diameters of the ILs [C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br are 330 ± 2 nm (PDI = 0.31), 454 ± 5 nm (PDI = 0.80) and 592 ± 4.6 nm (PDI = 0.29), respectively, which increased up to 456 ± 7 (PDI = 0.66), 560 ± 3 (PDI = 0.51), and 967 ± 5.2 nm (PDI = 0.6) due to the incremental addition of β-CD. Thus, the particle size enhancement and broader particle size distribution spectra in a higher concentration of β-CD designate the supramolecular constructions through the intermolecular inclusion of the ILs into the hydrophobic cavity of CD.^55,56

3.14. Antibacterial Activities of ILs and Their Corresponding ICs

To investigate the antibacterial activity, all three ILs ([C₆CFBim]Br, [C₈CFBim]Br, and [C₁₀CFBim]Br) were treated against both Gram-positive (Bacillus subtillis CCM 4062) and Gram-negative (Escherichia coli K12) bacteria by Standard Bauer-Kirby disk tests.^57,58 Additionally, the role of complexation on the antibacterial activity of all the corresponding ICs also has been studied against the above-mentioned bacteria (Figure S12).

It has been found that all the ILs and their corresponding ICs show a microbicidal effect on both Gram-negative and Gram-positive bacteria at a very high dose (MIC99 dose of 25 mM and 2.5 mM, respectively) where the latter is more effectively targeted. The killing effectiveness was calculated by persistent colony forming units (cfu) of bacteria that sustained drug treatment (Table S8).

The observation concluded that ILs follow the order viz. [C₆CFBim]Br ∼ [C₁₀CFBim]Br > [C₈CFBim]Br for Gram-positive bacteria and [C₆CFBim]Br > [C₁₀CFBim]Br > [C₈CFBim]Br for Gram-negative bacteria (Figure S13).

Here, the antibacterial activity of IL showed little deviation in antibacterial activity from imidazolium ILs, where in case of imidazolium ILs antibacterial activity increased with the increase of alkyl chain length.⁵⁹ This can be explained as the optimum biological effect at a specific chain length depends on several physicochemical parameters: surface activity, adsorption, transport in the test medium, aqueous solubility, and hydrophobicity.²³

However, when there is a comparison of antibacterial activity between the ILs and the corresponding ICs, it is very obvious that hydrophobic interaction between the hydrophobic long-alkyl tail of the ILs and the lipid membrane of bacteria is lesser when it forms ICs than that of the free ILs. Hence, it should show that there is lesser antibacterial activity (occurred at B6 and B10) than that of free ILs. In contrast, from Figure S13, it is clear that the antibacterial activity of B8 is more than [C₈CFBim]Br possibly because of the optimal biological activity at this specific chain length and other factors such as distinct hydrophobicity, water miscibility, and absorptivity on the test medium.²³ Consequently, that may proficiently compel the ICs to target the bacterial membrane and therefore increase their antibacterial activity.²²

MIC values (Table S8) as well as the zone of inhibition (Table S9 and Figure S12 and Figure S13), determined for the tested drug candidates, were very high in comparison to the antibiotics prescribed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). It may be inferred that as-synthesized ILs and ICs have the least role as an antibacterial agent.

3.15. Mechanism of Antibacterial Activity

For attaining visual understanding of the antibacterial activities of the ILs and ICs, the morphological study has been carried out by SEM to understand the response of the bacterial cell membrane when it is exposed in the presence of ILs and their corresponding ICs (Figure 10). The bacteria from the microtiter plate (used in MIC test) have been used for scanning electron microscopy (SEM). Both E. coli and B. subtilis from the wells of the microtiter plate where their growth was inhibited were included. Bacteria without any additives (drug/IL) were taken as the negative control wells whereas bacteria with Ampicillin were taken as positive control wells. For SEM study of the bacteria a standardized protocol was used (Figure 10).⁶⁰

Figure 10.

SEM images of E. coli (left) and B. subtillis (right) under (a) −ve and (b) +ve control.

From the comparative SEM images of the bacteria under drug (ILs and ICs) treatment and bacteria under the negative control (Figure 10, Figure 11, and Figure S14), it is clear that the morphological changes of the bacteria took place due to the drug’s effect at a very high dose. The average length of the bacteria E. coli is approximately 2 μm under normal growth conditions, and B. subtilis is approximately 3–10 μm under normal growth conditions. It has been observed that there is a visible shrinkage in the cell wall of bacteria under the influence of the drugs (ILs and ICs).

Figure 11.

SEM images of E. coli incubated in the presence of different ILs and ICs (a) ([C₆CFBim]Br and B6 (10 mg/mL)), (b) ([C₈CFBim]Br and B8 (10 mg/mL)), and (c) ([C₁₀CFBim]Br and B10 (10 mg/mL)) at their different concentrations.

Hence, we have hypothesized that the ILs form electrostatic attractions between the cationic headgroup and bacterial cell membrane, which contains 70% negative charge containing phosphatidylethanolamine. Subsequently, ILs disrupt the inner cell membrane by forming hydrophobic interaction between the hydrophobic tail and the hydrophobic phospholipids part of the inner cell membrane. Finally, they lead to the leakage of cytoplast. It is very difficult for bacteria to reconstruct the physically impaired cell wall; consequently, bacteria get killed by the ILs.⁶¹

4. Conclusions

In summary, studied benzimidazolium ILs show a 1:1 inclusion complex formation which is confirmed by HRMS, surface tension, conductivity, and ITC. P-XRD analysis represents that the ICs are isomorphic and possess a channel-like arrangement, which adopts a symmetrical conformation with the β-CD. TGA-DSC indicate as prepared ICs have lower thermal stability than that of host and guest in each case; probably geometry distortion and steric congestion of β-CD lower the thermal stability of ICs. The highest chemical shift obtained from NMR, the broad nature in IR spectra, and the maximum binding constant obtained from ITC prove that [C₁₀CFBim]Br comes from the strongest IC with β-CD. Thus, B10 would be a better candidate to show controlled delivery when forming ICs with β-CD. Antibacterial study follows the order as [C₆CFBim]Br > [C₁₀CFBim]Br > [C₈CFBim]Br which shows a deviation from imidazolium ILs. ICs of [C₆CFBim]Br and [C₁₀CFBim]Br, i.e. B6 and B10, show lower antibacterial activity than that of the corresponding ILs may be because of the reduced hydrophobicity of IL when forming ICs. On the other hand, the specific chain length and distinct hydrophobicity make B8 more active than [C₈CFBim]Br to target the surface of bacteria. Further, results obtained in this work will encourage researchers to explore benzimidazole IL–CD conjugate as a potential candidate in controlled drug delivery and other biomedical applications.

Glossary

Abbreviations

ILs

ionic liquids

IC

inclusion complex

β-CD

beta cyclodextrin

CFBILs

carboxy functionalized benzimidazolium ILs

[C6CFBim]Br

3-(2-carboxy ethyl)-1-hexyl-1H-benzo[d]imidazol-3-ium bromide

[C8CFBim]Br

3-(2-carboxy ethyl)-1-octyl-1H-benzo[d]imidazol-3-ium bromide

[C10CFBim]Br

3-(2-carboxy ethyl)-1-decyl-1H-benzo[d]imidazol-3-ium bromide

B6

3-(2-carboxy ethyl)-1-hexyl-1H-benzo[d]imidazol-3-ium bromide + β-CD

B8

3-(2-carboxy ethyl)-1-octyl-1H-benzo[d]imidazol-3-ium bromide + β-CD

B10

3-(2-carboxy ethyl)-1-decyl-1H-benzo[d]imidazol-3-ium bromide + β-CD

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsphyschemau.1c00016.

• ¹H NMR, ¹³C NMR, and ESI-MS spectra of ILs, photographs of ICs, ¹H NMR, 2D-ROESY NMR, vibrational frequencies table for all ILs and ICs, ESI-MS spectra of ICs, measurements of specific conductance and surface tension for ICs, SEM images and TGA of ILs and ICs, DSC of ICs, photographs of zone of inhibition tests, SEM images of E. coli and B. subtillis under −ve and +ve control (PDF)

The authors declare no competing financial interest.

Notes

This work has been partly presented in CRSI NSC-24 (The 24th CRSI National Symposium in Chemistry (Feb. 8–10, 2019), CSIR-CLRI, India and MEDCHEM–2019 (Nov. 1–2, 2019), IIT Madras, India and received ‘ACS Best Poster Presentation Award’ in CRSI NSC-24.