Insights into the Interactions of Sulfamethoxazole with Organized

Nov 1, 2018 - Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (E), ... Institute of Forensic Science & Criminology, Panjab Unive...
0 downloads 0 Views 3MB Size
Download PDF
Article Cite This: Langmuir 2018, 34, 14624−14632

pubs.acs.org/Langmuir

Insights into the Interactions of Sulfamethoxazole with Organized Assemblies of Ionic and Nonionic Surfactants Aparna Saraf,† Shweta Sharma,‡ and Shilpee Sachar*,† †

Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400098, India Institute of Forensic Science & Criminology, Panjab University, Chandigarh 160 014, India

‡

Downloaded via YORK UNIV on December 7, 2018 at 11:17:14 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: This work reports the physicochemical behavior of antibiotic drug sulfamethoxazole (SMX) in the presence of different surfactants, viz., cetyl trimethyl ammonium bromide (CTAB), dodecyl trimethyl ammonium bromide, didodecyl dimethyl ammonium bromide, sodium dodecyl sulfate, sodium deoxycholate, Tween 80, and Tween 20. The drug−surfactant systems were studied by UV−visible and fluorescence spectroscopies to assess the binding constants (Kb), partition coefficient (Kx), free energy of partition (ΔGp), aggregation number (Nagg), and quenching constant (KSV). Solubilization studies were carried out to understand the encapsulation efficiency of the system, which was found to increase as a function of CTAB concentration. Surface tension measurements enabled us to determine the change in critical micelle concentration as well as to calculate the variation in surface parameters of surfactant in the presence of drug, viz., surface pressure (π), surface excess concentration (γmax), and minimum area (Amin). In addition, UV−visible, fluorescence, and circular dichroism studies were carried out to check the effects of surfactant-based SMX formulation on serum proteins.

■

INTRODUCTION Rational drug designing aims toward a formulation with minimum drug degradation, enhanced dissolution, and targeted action along with enhanced bioavailability of the drug. Most of the promising drugs from the new generation of antibiotics suffer from low aqueous solubility, low penetration, poor bioavailability, low stability, and hence low therapeutic efficacy. This along with an increasing frequency of antibioticresistant bacteria has become a challenge for medicinal chemists. In addition, with natural product screening and chemical synthetic routes for a new antibiotic moiety, attempts should also be made for improving the efficacy of existing promising antibiotics by choosing different excipients such as surfactants, sugars, and nanoparticles in association with the desired drug in order to develop effective formulations which can facilitate the therapeutic action. Surfactant micelles are found to facilitate drug absorption along with shielding of the active drug molecules from adverse environmental conditions. The study on molecular-level interactions between the drug and micelles can be used to predict several pharmacokinetic and pharmacological properties of drugs, viz., transport, biodistribution, accumulations, and therefore their efficacy. When a sufficient amount of a surfactant is dissolved in water, the surfactant molecules form colloidal clusters (micelles) of various shapes in which the polar head groups point outward and the hydrophobic ends point toward the core © 2018 American Chemical Society

of the micelle. The threshold concentration at which the formation of micelle begins is known as critical micelle concentration (cmc). Micellar core is capable of incorporating hydrophobic substances present in the system. In past, various ionic surfactants such as cetyl trimethyl ammonium bromide (CTAB), dodecyl trimethyl ammonium bromide (DTAB), tetradecyl trimethyl ammonium bromide, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, alpha olefin sulfonate, alkylbenzene sulfonate alone, and their mixtures have been studied in association with various poorly water-soluble drugs, viz., ibuprofen,1,2 naringenin,3 danazol,4 gliclazide,5 and so forth. Nonionic surfactants, viz., Brij 351 and Tween 80,5 have also been studied in association with poorly soluble drugs for enhancing their solubility. Sulfamethoxazole (SMX) (Figure 1) belonging to class sulfonamides is an Food and Drug Administration approved

Figure 1. Molecular structure of SMX. Received: August 18, 2018 Revised: October 27, 2018 Published: November 1, 2018 14624

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir

All the chemicals were of analytical grade and used without further purifications. The experiments were performed in phosphate buffer (pH 7.4). Double-distilled water was used for the entire study. UV−Visible Studies. The effects of various types of surfactants on the absorption spectrum of SMX were studied using a UV-2450 Shimadzu UV−vis spectrophotometer with quartz cuvettes of 1 cm path length at 298 K in the wavelength range of 200−800 nm. The concentration of surfactants was varied from pre- to postmicellar concentration, viz., 0.1−20 mM for CTAB (cmcaq = 0.98 mM), 1−25 mM for DTAB (cmcaq = 14 mM), 0.01−1 mM for DDAB (cmcaq = 0.08 mM), 0.001−1 mM for Tween 80 (cmcaq = 0.023 mM), 0.01− 0.2 mM for Tween 20 (cmcaq = 0.064 mM), 1−20 mM for SDS (cmcaq = 8.6 mM), and 1−10 mM for NaDOC (cmcaq = 2 mM). In all the experiments, the concentration of SMX was kept constant at 0.1 mM. Entire experiments were performed in phosphate buffer (pH 7.4). Each set was carried out in triplicate, and standard deviations are represented by the error bars in the graphs. Fluorescence Studies. All the fluorescence experiments were performed on a RF-5301PC Shimadzu spectrofluorophotometer at 298 K. The experiments were performed with a quartz cell of 1 cm path length. For fluorescence measurements, the concentration of pyrene was kept constant at 1 mM. SMX concentration was varied from 0.02 to 0.2 mM, and the concentration of surfactants was kept constant at 20 mM for CTAB, 25 mM for DTAB, and 1 mM for Tween 80. The excitation wavelength was selected at 335 nm to selectively excite pyrene probe, and corresponding emission spectra were recorded in the range of 345−600 nm. Excitation and emission slit widths were fixed at 5 nm. Each set was carried out in triplicate, and standard deviations are represented by the error bars in the graphs. Protein Interactions. Absorption spectra and fluorescence quenching studies of BSA (1 mg/mL) were recorded in the presence of increasing concentration of SMX (0−0.1 mM) with the constant concentration of CTAB (20 mM), DTAB (25 mM), and Tween 80 (1 mM). For the fluorescence analysis, the excitation and emission slit width were fixed at 5 nm in the wavelength range of 287−500 nm with the excitation wavelength at 277 nm. Each set was carried out in triplicate, and standard deviations are represented by the error bars in the graphs. CD spectra in the far UV CD (190−260 nm) and near UV CD (260−360 nm) regions were obtained on a JASCO J-815 CD spectrometer at 298 K for observing the alterations in the secondary and tertiary structures of the serum protein (BSA). Spectropolarimeter was thoroughly purged with N2 gas before starting the experiment. Each spectrum was baseline corrected and was taken as an average of three accumulations at a scan rate of 100 nm/min with a response time of 1 s. The secondary and tertiary structures of BSA were observed in the absence and presence of SMX, CTAB, and Tween 80 using cuvettes of 1 cm path length. SMX concentration was kept constant at 0.1 mM, and CTAB, DTAB, and Tween 80 concentrations were kept constant at 20, 25, and 0.1 mM, respectively, for the CD studies. The concentration of BSA used was 0.3 × 10−6 mol dm−3 for far UV and 15 × 10−6 mol dm−3 for near UV CD experiments. The molar ellipticity was calculated and plotted against the wavelength. To check the structural changes in BSA, the study was carried out for 48 h. Solubility Measurement. Phase solubility studies of SMX in CTAB were carried out according to the Higuchi−Connors procedure.15 SMX (0.1 g) was placed in each flask containing different concentrations (pre to post) of CTAB in phosphate buffer (pH 7.4). The flasks were stirred at 298 K for 24 h to achieve the equilibrium. Appropriate aliquots were then withdrawn, filtered, and subsequently diluted with buffer (pH 7.4). The total concentration of the drug in the filtrate was analyzed by UV absorbance studies. A control assay was performed in the absence of surfactant, which was used for the construction of a calibration curve. Each set was carried out in triplicate. Standard deviations are represented by the error bars in the graphs. Surface Tension Studies. Interfacial tension was measured by pendant drop method using a 260-F4 rame-hart goniometer at 298 K.

drug, which is one of the most frequently used antibiotics in combination with trimethoprim (TMP). It is found in the blood as the protein-bound, unbound, metabolized form and as the conjugated form, viz., N-glucuronide conjugate. The free form of SMX is found to be the therapeutically active form. At pH values above 5.6, it is predominantly anionic; below 1.7, it is positively charged; and in between, it is uncharged. The drug is able to combine with the bacterial cell and hence efficient in inactivating toxic proteins, but its poor aqueous solubility results in severe side effects and hence limits its usage. Studies have been reported in the past to improve its therapeutic index alone6 and along with TMP7 via complexation approach using metal complexes,6 cyclodextrins,8,9 and surfactants.9 SMX showed a higher affinity toward β-CD and HP β-CD, and TMP showed a higher tendency of complexation with γ-CD. Also, the solubility of TMP showed higher enhancement than that of SMX in the presence of SDS surfactant. Its association with cationic benzalkonium chloride surfactants has also been studied using 1H NMR and conductivity measurements.10 SMX was found to be solubilized in the outer micellar portions. Gemini surfactants have also been studied in association with SMX via adsorption isotherms, mainly to study the extent of SMX adsorption on micellar surface for effective removal of antibiotics from wastewater.11 SMX alone and in combination with TMP has also been studied in association with nanoparticles for developing a targeted delivery formulation.12,13 Reports suggested successful loading of SMX on nanomodalities via different protocols. To investigate its fate in vivo, interactions of SMX along with sulfanilamide were studied with serum proteins and nucleobases using molecular docking investigation and UV− visible and fluorescence studies. 14 Very few micellar formulations have been studied in association with SMX, and thus, because of the paucity of information, the present study focuses on studying SMX with various classes of surfactants in order to find a suitable micellar core for SMX solubilization. Micellar solubilization of drugs can arise due to hydrophobic and/or electrostatic interactions. Surfactants from ionic [CTAB, DTAB, didodecyl dimethyl ammonium bromide (DDAB), SDS, and sodium deoxycholate (NaDOC)] and nonionic classes (Tween 80 and Tween 20) were comprehensively studied in association with SMX via UV−visible, fluorescence, and surface tension measurements. Being the major transport proteins, serum albumins are widely investigated with administered therapeutic formulations. Drug−protein interactions not only affect drug distribution, but can bring conformational changes in proteins which can trigger toxicity and hence affect the in vivo fate of the formulations. Circular dichroism (CD) studies were carried out to study conformational changes in bovine serum albumin (BSA) in the presence of SMX−surfactant systems. The study provides insights into the association of SMX with different surfactants (CTAB, DTAB, DDAB, SDS, NaDOC, Tween 80, and Tween 20). The findings would, in turn, serve as an integral knowledge for the design of more competent surfactant-based drug formulations for SMX with improved prolonged efficacy of the drug.

■

MATERIALS AND METHODS

Materials. SMX, fatty-acid-free BSA, CTAB, DTAB, DDAB, SDS, NaDOC, Tween 80, Tween 20, sodium phosphate dibasic, and sodium phosphate monobasic with purity ≥98% were procured from Sigma-Aldrich. Pyrene with 98% purity was obtained from Alfa Aesar. 14625

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir

Figure 2. UV spectrum of [a] SMX (0.1 mM) in the presence of varying concentrations of CTAB (0.1−20 mM), inset: Benesi−Hildebrand plot of the SMX−CTAB system; [b] SMX (0.1 mM) in the presence of varying concentrations of DTAB (1−25 mM), inset: Benesi−Hildebrand plot of the SMX−DTAB system; [c] SMX (0.1 mM) in the presence of varying concentrations of Tween 80 (0.001−1 mM), inset: Benesi−Hildebrand plot of the SMX−Tween 80 system; and [d] SMX (0.1 mM) in the presence of varying concentrations of SDS (1−20 mM) in phosphate buffer (pH 7.4) at 298 K.

concentration of surfactant, ΔA is the differential absorbance, and Kb is the binding constant. Binding constant was found to be maximum for the SMX− Tween 80 system [(1.88 ± 0.1) × 104 M−1] in comparison to SMX−CTAB [(0.12 ± 0.03) × 104 M−1] and SMX−DTAB [(0.01 ± 0.001) × 104 M−1] [Table S1]. These values are in accordance with other drug−surfactant systems studied in the past.17 Partition Coefficient. The partition coefficient (Kx) of SMX was evaluated between water and micellar pseudo-phase. It is the ratio of the concentration of drug molecules in the micelle to that in the bulk aqueous solution. The partition coefficient parameter is important not only in elucidating the mechanism of solubilization, but also helps to understand how a drug is partitioned through biological membranes within the living body. The partition coefficient was obtained from the following equation:18,19

cmc and surface properties of CTAB were determined in the absence and in the presence of a constant concentration of SMX (0.1 mM) in phosphate buffer (pH 7.4). Each set was carried out in triplicate, and standard deviations are represented by the error bars in the graphs.

■

RESULTS AND DISCUSSION Electronic Absorption Studies. Figure S1a shows the absorption spectrum of varying concentrations of SMX in pH 7.4. It indicates absorption maxima at 256 nm with a molar extinction coefficient of 31 755 mol−1 dm3 cm−1 (R2: 0.9998) obtained from the plot of absorbance versus SMX concentration [inset of Figure S1a]. Figure 2a shows the absorption spectra of SMX in the presence of varying concentrations of CTAB. It was observed that as the surfactant concentration increases, the absorbance increases slightly with the shift in maxima from 256 to 260 nm. This observed red shift [inset of Figure 2a] and the increase in absorbance clearly indicates the interaction between negatively charged SMX and positively charged CTAB moieties. The wavelength shift was found to be maximum for CTAB in comparison to DTAB [inset of Figure 2b]. A slight increase in absorbance with no shift in wavelength was observed in the presence of Tween 80 [Figure 2c]. No significant changes were observed in the presence of SDS [Figure 2d], DDAB, NaDOC, and Tween 20 based systems [Figure S1b−d], which point toward very weak interactions. Thus, the extent of binding between SMX−CTAB/DTAB/ Tween 80 was evaluated using Benesi−Hildebrand equation:16 1 1 1 = + ΔA At − A0 Kb[S](A t − A 0)

nw 1 1 = + ΔA ΔA∞ K x ΔA∞([S] + C t − cmc)

(2)

where ΔA = A − A0 and ΔA∞ = Ab − A0. A, A0, and Ab are the absorbance values of SMX in the presence of surfactants, in the absence of surfactants, and the absorbance because of the formation of drug−surfactant complex, respectively; CT is the total concentration of SMX; [S] is the concentration of surfactant; nw is the molarity of water (55.5 M); and Kx is the partition coefficient. The plots of 1/ΔA versus 1/([S] + Ct − cmc) are shown in Figure S2a−c for SMX−CTAB, SMX−DTAB, and SMX−Tween 80 systems. The values for Kx were found to be 3.29 (±0.2) × 106, 2.14 (±0.3) × 105, and 1.44 (±0.1) × 104 for the SMX−CTAB, SMX−Tween 80, and SMX−DTAB systems, respectively

(1)

where A and A0 are the values of absorbance of SMX in the presence and absence of surfactants, respectively, At is the absorbance of SMX bound to surfactant micelles, [S] is the 14626

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir

Figure 3. Fluorescence spectra of [a] pyrene (1 mM) in the presence of CTAB (20 mM) at varying concentrations of SMX (0.02−0.2 mM), inset: plot of ln F0/F vs [Q]; [b] pyrene (1 mM) in the presence of DTAB (25 mM) at varying concentrations of SMX (0.02−0.2 mM), inset: plot of ln F0/F vs [Q]; and [c] pyrene (1 mM) in the presence of Tween 80 at varying concentrations of SMX (0.02−0.2 mM) in phosphate buffer (pH 7.4) at 298 K.

(Table S1), which is in accordance with the literature values for drug−surfactant systems.19 The drug partitioned between the micellar and water phases as a result of the hydrophobic or electrostatic interactions. The high positive value for the SMX−CTAB system in comparison to the SMX−Tween 80 and SMX−DTAB systems suggests that the electrostatic interaction plays a major role along with the hydrophobic interactions, as under working conditions, SMX is negatively charged. This drives negatively charged SMX to partition or solubilize more in the CTAB micelles. The standard free-energy change for the transfer of SMX from water to micellar phase (ΔG0x ) was evaluated using the following equation: ΔGx0 = −RT ln K x

ln

I0 [Q] = I [M]

(4)

where [M] =

[S]T − cmc Nagg

(5)

where [M] here represents the micellar concentration and [Q] is the quencher concentration, I represents the intensities in the presence and I0 represents the intensities in the absence of quencher, [S]T is the concentration of surfactant, and Nagg is the micellar aggregation number. The slope of eq 4 is used to calculate the aggregation number [Figure S3a,b]. The aggregation numbers for the SMX−CTAB and SMX−DTAB systems were found to be 61 (±1) and 54 (±2), respectively (Table S1). The decrease in fluorescence intensity with an increasing SMX concentration strongly points toward the electrostatic interactions. The values of aggregation number show a minor increase in the case of SMX−CTAB/DTAB systems in comparison to the aggregation number of surfactants alone.22,23 This can be visualized as the presence of SMX on the palisade layer of the surfactant that results in more compact and stable aggregates as the hydrophobic part of the drug is distributed toward the nonpolar part of CTAB, whereas the ionic part of the drug interacts with the head group of the surfactant. No significant quenching was observed for SMX−Tween 80 system and hence Nagg was not calculated. The quenching constant for SMX−CTAB and SMX−DTAB systems was evaluated using the Stern−Volmer equation:

(3)

where R is the universal gas constant and T is the temperature. ΔGx for the systems SMX−CTAB, SMX−Tween 80, and SMX−DTAB was found to be −33, −27, and −21 kJ mol−1, respectively. The negative value indicates that SMX partitioning between the micellar and the bulk water phases happens spontaneously. Fluorescence Studies. The molecular association and dynamics between SMX and surfactants were monitored by changes in the fluorescence emission of the probe (pyrene) as SMX is a very weak fluorescent drug. The relative intensity of I and III emission bands of pyrene is related to environment polarity and thus can give specific information on the absence or presence of micellar or other hydrophobic domains.20 With increasing concentrations of SMX in the presence of CTAB, DTAB, and Tween 80 [Figure 3a−c], a decrease in I1 and I3 peaks of pyrene emission spectra was observed. This indicates that the environment surrounding the pyrene gets changed, which is a result of the association of quencher (SMX) with the micelles. The aggregation numbers were also determined from the fluorescence results using the following equation:21

F0 = 1 + KSV[Q] F

(6)

where F represents the fluorescence intensities in the presence and F0 represents the intensities in the absence of quencher 14627

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir

Figure 4. UV spectrum of [a] BSA in the presence of CTAB (20 mM) at varying concentrations of SMX (0.02−0.1 mM), inset: Benesi− Hildebrand plot; [b] BSA in the presence of DTAB (25 mM) at varying concentrations of SMX (0.02−0.1 mM), inset: Benesi−Hildebrand plot; [c] BSA in the presence of Tween 80 (1 mM) at varying concentrations of SMX (0.02−0.1 mM), inset: Benesi−Hildebrand plot in phosphate buffer (pH 7.4) at 298 K.

Figure 5. Fluorescence spectra of [a] BSA in the presence of CTAB (20 mM) at varying concentrations of SMX (0.02−0.1 mM), inset: modified Stern−Volmer plot; [b] BSA in the presence of DTAB (25 mM) at varying concentrations of SMX (0.02−0.1 mM), inset: modified Stern−Volmer plot; [c] BSA in the presence of Tween 80 (1 mM) at varying concentrations of SMX (0.02−0.1 mM), inset: modified Stern−Volmer plot in phosphate buffer (pH 7.4) at 298 K.

values of KSV were found to be 4.53 (±0.002) × 103 and 6.87 (±0.02) × 103 M−1 for SMX−CTAB and SMX−DTAB systems, respectively (Table S1). Protein Interactions. UV−Vis Studies. The increase in the concentration of SMX causes enhancement in the absorbance of BSA with a blue shift in wavelength (from 277 to 261 nm)

concentration [Q]. KSV is the Stern−Volmer quenching constant which assesses the bimolecular quenching and decay as it is the product of rate constant of the quenching process and lifetime of the probe in the absence of quenching. Plots of F0/F versus [Q] for SMX−CTAB and SMX−DTAB systems are shown in the inset of Figure 3a,b, respectively. The 14628

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir

Figure 6. [a] Far UV, [b] near UV CD spectrum of protein in the presence of SMX (0.1 mM) at a constant concentration of CTAB (20 mM); [c] far UV and [d] near UV CD spectrum of protein in the presence of SMX (0.1 mM) at a constant concentration of DTAB (25 mM); and [e] far UV and [f] near UV CD spectrum of protein in the presence of SMX (0.1 mM) at a constant concentration of Tween 80 (0.1 mM) in phosphate buffer (pH 7.4) at 298 K.

fluorescence. In the BSA−SMX−Tween 80 [Figure 5c] system, quenching was observed without a significant shift in the emission wavelength of BSA. The fluorescence results obtained were analyzed using the Stern−Volmer relation (eq 6). Plots of F0/F versus [Q] shown slightly upward curvature; hence, the data were further analyzed by employing the modified Stern−Volmer equation given below:24

in the BSA−SMX−CTAB system [Figure 4a], BSA−SMX− DTAB system [Figure 4b], and BSA−SMX−Tween 80 system [Figure 4c]. This may be due to the formation of SMX−BSA ground-state complex in the presence of CTAB, DTAB, and Tween 80, which points toward the decrease in polarity around tryptophan residues that again confirms binding between SMX−CTAB/DTAB/Tween 80 and BSA systems. The extent of binding between BSA−SMX−CTAB/DTAB/ Tween 80 was evaluated using the Benesi−Hildebrand relation (eq 1). Plots of 1/ΔA vs 1/[S] for BSA−SMX−CTAB, BSA− SMX−DTAB, and BSA−SMX−Tween 80 systems are shown in the inset of Figure 4a−c, respectively. Binding constant was found to be maximum for the BSA−SMX−CTAB system [(1.28 ± 0.001) × 102 M−1] in comparison to the BSA− SMX−DTAB [(1.18 ± 0.03) × 102 M−1] and BSA−SMX− Tween 80 [(0.92 ± 0.003) × 102 M−1] systems (Table S2). Fluorescence Studies. The intrinsic fluorescence of BSA is essentially due to tryptophan, which is sensitive to any polarity change in the microenvironment. Fluorescence of BSA gradually decreased with the increasing concentration of SMX in the presence of CTAB [Figure 5a] and DTAB [Figure 5b]. A significant shift in the emission maximum wavelength (343− 334 nm) was also observed, indicating the SMX−BSA interaction that also supports quenching of intrinsic

F0 1 1 = + F0 − F fa KSV[Q] fa

(7)

where F and F0 are the fluorescence intensities of the protein in the presence and absence of SMX, respectively; KSV is the modified Stern−Volmer quenching constant, [Q] is the concentration of the quencher, and fa is the fraction of accessible tryptophan. The plot of F0/(F0 − F) versus 1/[Q] [inset in Figure 5a−c] indicates a linear trend. The value of fa was found to be 1.11 for BSA−SMX−CTAB, 0.96 for BSA− SMX−DTAB, and 1.14 for BSA−SMX−Tween 80. The corresponding KSV values are 30.0 (±0.003), 28.65 (±0.09), and 18.93 (±0.001) M−1, respectively (Table S2). Analysis of Binding Equilibria. If it is assumed that there are similar and independent binding sites in the biomolecule, for static quenching interaction, the binding constant, K, and 14629

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir

Figure 7. (a) Solubility curve and (b) EE of SMX as a function of CTAB concentration in phosphate buffer (pH 7.4) at 298 K.

Micellar Solubilization of SMX in CTAB. From the UV− visible and fluorescence spectroscopic observations, it was found that interactions between SMX and cationic surfactants are predominantly dominant. As changes in the near UV CD spectra were observed in the DTAB system, thus, further studies were carried out only for SMX−CTAB systems. Figure 6 shows the solubility change of SMX as a function of CTAB concentration, which indicates that, above the cmc, the solubility of the drug increased with the increase in surfactant concentration. The solubilization effect of CTAB on SMX is stronger, and this behavior is a consequence of the electrostatic interactions between the positively charged surfactant CTAB and anionic drug SMX, along with the hydrophobic interactions. Drug encapsulation efficiency (EE, %) was determined from solubility measurements, based on the weight ratio of the amount of SMX in micelles to the initial amount of drug added.28

the number of binding sites, n, can be calculated using the equation25 log

F0 − F = log K + n log[Q] F

(8)

From the linear plot of log (F0 − F)/F versus log[Q], the values of K and n were obtained from the intercept and slope, respectively [Figure S4a−c] (Table S2). Values of K for the BSA−SMX−CTAB, BSA−SMX−DTAB, and BSA−SMX− Tween 80 systems were found to be 6.60 (±0.01) × 105, 1.34 (±0.01) × 103, and 3.09 (±0.02) × 105 M−1, respectively. n value for the BSA−SMX−CTAB system was 1.3, which is greater than BSA−SMX−DTAB (0.9) and BSA−SMX−Tween 80 (1.2) systems. From the values of n, it may be inferred that there is one independent binding site on BSA for SMX in the presence of CTAB, DTAB, and Tween 80. The value of ΔG0 was calculated using the equation given below: ΔG 0 = −2.303RT log K

Encapsulation efficiency =

(9)

ΔG0 value for the BSA−SMX−CTAB system BSA−SMX− DTAB and BSA−SMX−Tween 80 systems was found to be −33.58, −18.07, and −31.68 kJ M−1, respectively. The negative values of ΔG0 indicate that the processes were spontaneous. Denaturation Studies. In the far UV region, native BSA showed two negative minima at 208 nm (corresponding to π → π* transition for α helix) and 222 nm (corresponding to n → π* transition for both α helix and random coil), which are characteristic of the α-helical structure of proteins.26,27 This replicates helical conformation of the protein, and any change in the α-helix can thus be seen via changes in the CD spectra. The near UV CD spectrum helps to detect the tertiary structural changes in protein due to unfolding. The secondary and tertiary structures of BSA were observed in the presence and absence of SMX, CTAB, DTAB, and Tween 80 [Figure 6a−f] systems via accompanying changes in molecular ellipticity in the far and near UV regions. BSA in the presence of SMX and Tween 80 shows stable secondary structure even after 48 h, whereas the presence of CTAB and DTAB along with the drug changes the secondary structure of the protein. These changes can be attributed to partial rupture of intrachain hydrophobic interactions between the polypeptides leading to the expanding of polypeptide and unfolding of BSA. The near UV spectrum indicates minor changes in the tertiary structures of BSA in the presence of DTAB after 48 h; however, the BSA structure was maintained in the presence of CTAB and Tween 80 even after 48 h and thus confirming long-term biocompatibility of these systems.

amount of SMX in micelles initial amount of SMX added (10)

EE of SMX was found to increase as a function of CTAB concentration as shown in Figure 7b. Interfacial Phenomenon. Surface tensiometry is an effective technique for studying the surface phenomenon of surfactants and macromolecules. The plot of surface tension (γ) versus log[C] in the presence and absence of SMX is given in Figure 8. The cmc of CTAB in buffer solution (pH 7.4) was found to be 1.0 × 10−3 mol dm−3. The cmc of the surfactant in the presence of SMX shifted toward a higher concentration

Figure 8. Variation of surface tension as a function of CTAB concentration in phosphate buffer (pH 7.4) at 298 K. 14630

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir (1.8 × 10−3 mol dm−3), which is an indication of the utilization of surfactant monomers in binding with SMX, which leads to delayed micellization. The slope of the tensiometric profile near the cmc is a measure of interfacial adsorption efficacy of the surfactant, which was quantified by the Gibbs surface excess (γmax)29−31 and is given by γmax = −

ij δγ yz 1 jj zz 2.303RTn jjk δ log C zz{

greater in the case of CTAB, DTAB, and Tween 80 systems at physiological pH 7.4. Large values of the partition coefficient for SMX in CTAB micelles [3.29 (±0.2) × 106] showed spontaneous partitioning of drug molecules from aqueous media to cationic CTAB micellar media in comparison to DTAB [1.44 (±0.1) × 104] and Tween 80 [2.14 (±0.3) × 105] systems. Another interesting point is that the SMX−CTAB system showed strongest binding with transport serum proteins (BSA), whereas weak interactions were observed for Tween80-based systems. Also, changes in the tertiary structure of BSA were observed in the presence of SMX−DTAB systems. CTAB-based SMX formulations are thus found to be stronger and long-term biocompatible. Solubility studies confirm the high solubility and increasing EE of SMX with increasing CTAB concentrations. Surface properties, viz., surface excess concentration γmax, minimum area per molecule Amin, and surface pressure πcmc, provide a better understanding about the location of SMX in CTAB micelles. The findings will contribute insights into understanding biomolecular interactions between surfactants with SMX, which can aid the design of a competent biocompatible SMX formulation. The future of such studies lies in understanding the interaction of a variety of drugs with different excipients quantitatively in order to gain insights into the functional groups responsible for interaction/partitioning and hence deriving guidelines for target-oriented synthesis of new drug formulations.

(11)

where γ is the surface tension of the solution, R is the universal gas constant (8.314 J K−1 mol−1), T is the temperature in the absolute scale, C is the concentration of the surfactant in solution, and n is a constant that depends on the number of species constituting the surfactant, and in the present case, it is equal to 1 because of the presence of high ionic strength of the buffer solution used. The minimum area of exclusion per molecule at the saturated air/solution interface (Amin) is given by A min =

1018 NAγmax

(12)

where NA is Avogadro’s number. The surface pressure at the cmc (πcmc) was obtained from the following equation: πcmc = γ0 − γcmc (13) where γ0 and γcmc are the surface tensions of pure solvent and at the cmc, respectively. The interfacial parameters of CTAB in the absence and presence of SMX are given in Table 1.

■

ASSOCIATED CONTENT

* Supporting Information S

Table 1. Values of γmax, πcmc, and cmc Evaluated from Surface Tension Measurements SMX (M)

cmc[CTAB] × 10−3 (M)

γmax × 106 (mol/m2)

Amin (nm2)

πcmc (mN m−1)

0 0.1 × 10−3

1.0 1.8

5.06 6.11

0.327 0.271

27.83 30.38

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b02814. UV spectra of [a] SMX (0.02−0.1 mM), inset: calibration graph of SMX, [b] SMX (0.1 mM) in the presence of varying concentrations of DDAB (0.01−1 mM), [c] SMX (0.1 mM) in the presence of varying concentrations of NaDOC (1−10 mM), and [d] SMX (0.1 mM) in the presence of varying concentrations of Tween 20 in (1−0.2 mM) phosphate buffer (pH 7.4) at 298 K; plot of 1/ΔA versus 1/([S] + Ct − cmc) for [a] SMX−CTAB system, [b] SMX−DTAB system, and [c] SMX−Tween 80 system; plot of ln I0/I versus [Q] for the aggregation number of [a] SMX−CTAB system and [b] SMX−DTAB system in phosphate buffer (pH 7.4) at 298 K; plot of log F0 − F/F versus log Q for the BSA at varying concentrations of SMX [a] in the presence of CTAB (20 mM), [b] in the presence of DTAB (25 mM), and [c] in the presence of Tween 80 (1 mM) in phosphate buffer (pH 7.4) at 298 K; values of binding constant (Kb), partition coefficient (Kx), aggregation number (Nagg), and quenching constant (KSV) for SMX−surfactant systems; and values of binding constant (Kb), quenching constant (KSV), equilibrium constant (K), and number of binding sites (n) for BSA−SMX− surfactant systems (PDF)

Lower surface tension for the SMX−CTAB system than that of CTAB alone indicates an interaction between SMX and CTAB, which ultimately leads to the increase in γmax. The higher γmax and hence lower Amin values obtained for the SMX−CTAB system compared to CTAB (in the absence of SMX) indicates a more compact monolayer. Strong electrostatic interactions along with the hydrophobic interactions create a pull for the negatively charged SMX moieties toward the micellar interface. This leads to the increase in surface excess concentration, which in turn decreases the area per molecule at the interface in the presence of SMX. This can be visualized as the closely packed SMX molecules with their orientation perpendicular to the interface. The increase in the surface pressure of CTAB (πcmc) in the presence of SMX again point toward the adsorption of SMX at the air/solution interface.

■

CONCLUSIONS The present work addresses the important need for a quantitative understanding of the solubilization and partitioning of the antibacterial drug SMX in different micellar media. The associations of CTAB, DTAB, DDAB, SDS, NaDOC, Tween 80, and Tween 20 were analyzed with SMX via UV− visible and fluorescence studies. The binding was found to be

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], dhamchem@gmail. com. Phone: +91-22-26543594. Fax: +91-22-26528547. 14631

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632

Article

Langmuir ORCID

Spectroscopic and molecular docking investigations. Spectrochim. Acta, Part A 2015, 144, 183−191. (15) Faustino, C.; Serafim, C.; Ferreira, I.; Pinheiro, L.; Calado, A. Solubilization power of an amino acid-based gemini surfactant towards the hydrophobic drug amphotericin B. Colloids Surf., A 2015, 480, 426−432. (16) Mahajan, S.; Mahajan, R. K. Interactions of phenothiazine drugs with bile salts: Micellization and binding studies. J. Colloid Interface Sci. 2012, 387, 194−204. (17) Usman, M.; Siddiq, M. Surface and micellar properties of Chloroquine Diphosphate and its interactions with surfactants and Human Serum Albumin. J. Chem. Thermodyn. 2013, 58, 359−366. (18) Enache, M.; Toader, A.; Enache, M. Mitoxantrone-Surfactant Interactions: A Physicochemical Overview. Molecules 2016, 21, 1356. (19) Enache, M.; Toader, A. M.; Neacsu, V.; Ionita, G.; Enache, M. I. Spectroscopic Investigation of the Interaction of the Anticancer Drug Mitoxantrone with Sodium Taurodeoxycholate (NaTDC) and Sodium Taurocholate (NaTC) Bile Salts. Molecules 2017, 22, 1079. (20) Mukhija, A.; Kishore, N. Drug partitioning in individual and mixed micelles and interaction with protein upon delivery form micellar media. J. Mol. Liq. 2018, 265, 1−15. (21) Azum, N.; Rub, M. A.; Asiri, A. M. Analysis of surface and bulk properties of amphiphilic drug ibuprofen and surfactant mixture in the absence and presence of electrolyte. Colloids Surf., B 2014, 121, 158− 164. (22) Pisárčik, M.; Devínsky, F.; Pupák, M. Determination of micelle aggregation numbers of alkyl trimethyl ammonium bromide and sodium dodecyl sulfate surfactants using time-resolved fluorescence quenching. Open Chem. 2015, 13, 922−931. (23) Stam, J. V.; Depaemelaere, S.; De Schryver, F. C. Micellar Aggregation Numbers - a Fluorescence Study. J. Chem. Educ. 1998, 75, 93−98. (24) Wang, G.; Wang, D.; Li, X.; Lu, Y. Exploring the binding mechanism of dihydropyrimidinones to human serum albumin: Spectroscopic and molecular modeling techniques. Colloids Surf., B 2011, 84, 272−279. (25) Shahabadi, N.; Hadidi, S.; Feizi, F. Study on the interaction of antiviral drug ’Tenofovir’ with human serum albumin by spectral and molecular modeling methods. Spectrochim. Acta, Part A 2015, 138, 169−175. (26) Joseph, D.; Sachar, S.; Kishore, N.; Chandra, S. Mechanistic insights into the interactions of magnetic nanoparticles with bovine serum albumin in presence of surfactants. Colloids Surf., B 2015, 135, 596−603. (27) Sandhya, B.; Hegde, A. H.; Kalanur, S. S.; Katrahalli, U.; Seetharamappa, J. Interaction of triprolidine hydrochloride with serum albumins: Thermodynamic and binding characteristics, and influence of site probes. J. Pharm. Biomed. Anal. 2011, 54, 1180− 1186. (28) Chhater, S.; Praveen, K. Solid Dispersion Incorporated Microcapsules: Predictive Tools for Improve the Half Life and Dissolution Rate of Pioglitazone Hydrochloride. Am. J. Biomed. Res. 2013, 1, 57−70. (29) Branco, M. A.; Pinheiro, L.; Faustino, C. Amino acid-based cationic gemini surfactant-protein interactions. Colloids Surf., A 2015, 480, 105−112. (30) Usman, M.; Rashid, M. A.; Mansha, A.; Siddiq, M. Thermodynamic solution properties of pefloxacin mesylate and its interactions with organized assemblies of anionic surfactant, sodium dodecyl sulphate. Thermochim. Acta 2013, 573, 18−24. (31) Ali, M. S.; Al-Lohedan, H. A. Interaction of biocompatible sugar based surfactant n-dodecyl β-d-maltoside with lysozyme. J. Mol. Liq. 2015, 209, 662−668.

Shweta Sharma: 0000-0002-1936-2947 Shilpee Sachar: 0000-0001-6207-3756 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge the financial support of the University Grant Commission in the terms of Non-NET Fellowship. This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

■

REFERENCES

(1) Jabbari, M.; Teymoori, F. An insight into effect of micelleforming surfactants on aqueous solubilization and octanol/water partition coefficient of the drugs gemfibrozil and ibuprofen. J. Mol. Liq. 2018, 262, 1−7. (2) Rangel-Yagui, C. O.; Hsu, H. W. L.; Pessoa, A., Jr.; Tavares, L. C. Micellar solubilization of ibuprofen − influence of surfactant head groups on the extent of solubilization. Braz. J. Pharm. Sci. 2005, 41, 237−246. (3) Jabbari, M.; Jabbari, A. Antioxidant potential and DPPH radical scavenging kinetics of water-insoluble flavonoid naringenin in aqueous solution of micelles. Colloids Surf., A 2016, 489, 392−399. (4) Vinarov, Z.; Katev, V.; Radeva, D.; Tcholakova, S.; Denkov, N. D. Micellar solubilization of poorly water-soluble drugs: effect of surfactant and solubilizate molecular structure. Drug Dev. Ind. Pharm. 2018, 44, 677−686. (5) Seedher, N.; Kanojia, M. Micellar Solubilization of Some Poorly Soluble Antidiabetic Drugs: A Technical Note. AAPS PharmSciTech 2008, 9, 431−436. (6) Mondelli, M.; Pavan, F.; de Souza, P. C.; Leite, C. Q.; Ellena, J.; Nascimento, O. R.; Facchin, G.; Torre, M. H. Study of a series of cobalt(II) sulfonamide complexes: Synthesis, spectroscopic characterization, and microbiological evaluation against M. tuberculosis. Crystal structure of [Co(sulfamethoxazole)2(H2O)2]·H2O. J. Mol. Struct. 2013, 1036, 180−187. (7) Markopoulou, C. K.; Malliou, E. T.; Koundourellis, J. E. Chemometric and derivative methods as flexible spectrophotometric approaches for dissolution and assaying tests in multicomponent tablets. Il Farmaco 2004, 59, 627−636. (8) Varghese, B.; Suliman, F. O.; Al-Hajri, A.; Al Bishri, N. S. S.; AlRwashda, N. Spectral and theoretical study on complexation of sulfamethoxazole with β- and HPβ-cyclodextrins in binary and ternary systems. Spectrochim. Acta, Part A 2018, 190, 392−401. (9) Göktürk, S.; Ç alışkan, E.; Talman, R. Y.; Var, U. A Study on Solubilization of Poorly Soluble Drugs by Cyclodextrins and Micelles: Complexation and Binding Characteristics of Sulfamethoxazole and Trimethoprim. Sci. World J. 2012, 2012, 1−12. (10) Farías, T.; de Ménorval, L. C.; Zajac, J.; Rivera, A. Solubilization of drugs by cationic surfactants micelles: Conductivity and 1H NMR experiments. Colloids Surf., A 2009, 345, 51−57. (11) Wang, J.; Gao, M.; Ding, F.; Shen, T. Organo-vermiculites modified by heating and gemini pyridinium surfactants: Preparation, characterization and sulfamethoxazole adsorption. Colloids Surf., A 2018, 546, 143−152. (12) Farías, T.; de Ménorval, L. C.; Zajac, J.; Rivera, A. Adsolubilization of drugs onto natural clinoptilolite modified by adsorption of cationic surfactants. Colloids Surf., B 2010, 76, 421−426. (13) Martínez-Costa, J. I.; Leyva-Ramos, R.; Padilla-Ortega, E.; Aragón-Piña, A.; Carrales-Alvarado, D. H. Antagonistic, synergistic and non-interactive competitive sorption of sulfamethoxazoletrimethoprim and sulfamethoxazole-cadmium (ii) on a hybrid clay nanosorbent. Sci. Total Environ. 2018, 640, 1241−1250. (14) Rajendiran, N.; Thulasidhasan, J. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: 14632

DOI: 10.1021/acs.langmuir.8b02814 Langmuir 2018, 34, 14624−14632