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Encompassment of Benzyl Isothiocyanate in cyclodextrin using ultrasonication methodology to enhance its stability for biological applications
Accepted Manuscript Encompassment of Benzyl Isothiocyanate in Cyclodextrin using Ultrasonication Methodology to enhance its stability for biological applications Shivani Uppal, Khushwinder Kaur, Rajendra Kumar, Nakshdeep Kaur Kahlon, Rachna Singh, S.K. Mehta PII: DOI: Reference:
S1350-4177(17)30164-5 http://dx.doi.org/10.1016/j.ultsonch.2017.04.007 ULTSON 3640
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
1 February 2017 4 April 2017 5 April 2017
Please cite this article as: S. Uppal, K. Kaur, R. Kumar, N.K. Kahlon, R. Singh, S.K. Mehta, Encompassment of Benzyl Isothiocyanate in Cyclodextrin using Ultrasonication Methodology to enhance its stability for biological applications, Ultrasonics Sonochemistry (2017), doi: http://dx.doi.org/10.1016/j.ultsonch.2017.04.007
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Encompassment of Benzyl Isothiocyanate in Cyclodextrin using Ultrasonication Methodology to enhance its stability for biological applications Shivani Uppala , Khushwinder Kaura*, Rajendra Kumarb, Nakshdeep Kaur Kahlonc, Rachna Singhc, S.K. Mehtaa* a
Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University, Chandigarh 160 014, India
b
UGC Centre of Excellence in Nanomaterials, Nanoparticles, Nanocomposites, Panjab University Chandigarh 160 014, India
c
Department of Microbial biotechnology, Panjab University Chandigarh 160 014, India
*Corresponding author: Khushwinder Kaur Tel: +919872040447; Fax: +91-172-2545074; Email: [email protected]
Abstract The use of methodical and innovative sonication method has been explored for the fabrication of inclusion complex of Benzyl Isothiocyanate, a potential anticancer and antimicrobial agent. The advancement involved investigation of inclusion behaviour, characterisation and an in-depth study of thermal and UV stability of Benzyl Isothiocyanate with cyclodextrins; β-CD and hp-β-CD. The sonication driven encompassment in cyclodextrins helped to overcome the hindrance of low solubility and high volatility. Investigations of physical and thermodynamic parameters using UV-visible spectroscopy, FTIR, XRD, TGA etc confirmed stability of inclusion complexes. Both β-CD and hp-β-CD based inclusion complexes retained the antimicrobial property of the free Benzyl Isothiocyanate, indicating their potential utility as antimicrobial agents. Haematological safety and cellular uptake data gives direction to in-depth analysis for its exploitation of antitumour activity.
Key words: cyclodextrin, benzyl isothiocyanate, thermal and UV stability, antimicrobial,
1. Introduction Benzyl isothiocyanate (BITC) is sulphur containing bioactive compound that exists predominantly in plants [1] like Tropaeolum mnajus L., Lepidium sativwm L., Carica papaya L. and Capparis flexuosa L. It shows excellent antibiotic activity against bacteria and fungi in vitro, possibly by targeting proteins and cell membrane integrity [2,3]. BITC induces cell cycle arrest, engender apoptosis and impede NFkB activation by triggering reactive oxygen species (ROS) [4,5]. The bioavailability of BITC in garden cress consumed by humans was approximately 14-50% due to the incapability of the sufficient therapeutic ingredient to be released from the vegetable by chewing so more than 50% of the delivered BITC is metabolized to its cysteine conjugate and ejected through urine [6]. Although BITC showed great potential preclinically but its effective impact during clinical investigation is impeded due to dearth of an adequate dosage form and poor bioavailability. β-Cyclodextrins (β-CD) and modified β-CDs are cyclic oligo glycosides having a polar, hydrophilic outside and a relatively nonpolar lipophilic inside [7]. The outer rim is hydrophilic, which is the reason behind the good solubility of CDs in aqueous media. Thus the solubility and stability of active guest-molecules gets amplified [8]. Low molecular weight compounds like BITC are best incorporated in β –CD [9]. Formation of inclusion complex (IC) of BITC is of enormous benefit in masking its acute odour, improving water solubility and meliorating its functional stability. In the present work, probe sonication, an extremely efficient, economical and time dipping technique capable of forming ICs [10] (reported in our previous work) has been used to form complexes of BITC with β-CD. Furthermore, complexation with modified β-CD and its detailed thermal studies have also been reported. The present report throws light on the enhanced stability of BITC in terms of thermodynamic parameters after complexation and its potential antimicrobial activity. This effortless, lucid and agile strategy not only produced the desirable outcomes quickly but also gave way to an environmental friendly methodology by avoiding the use of toxic solvents and saving endless labour. Further, both β-CD and modified β-CD (hp- β-CD) were investigated for haemato-compatibility as this interaction is expected after in vivo administration. Moreover, ICs loaded with BITC were able to permeate in vitro growing cancer cells equivalent to bare BITC. The cellular uptake behaviour was not affected by either of the ICs.
2. Experimental set-up and methodology 2.1. Materials Benzyl Isothiocyanate (> 99%), β-CD, (> 99%), hp-β-CD, (> 99%), Ethanol, (99%) and Hexane (> 99%) were procured from Sigma-Aldrich, India. MDA-MB-231 cells were used for cell uptake studies. Peripheral human blood from healthy volunteer was extracted to isolate the haematocrit and mononuclear cells for haematological studies. Triple distilled water was utilized to prepare samples. The structures of BITC and the two CDs used are reported in Scheme S1 (Supporting Information) 2.2. Preparation of Inclusion Complex Li et al. [11] reported the preparation of IC of BITC with β-CD by magnetic stirring followed by freeze drying ultrasonic homogenization for 3 h at 40ºC. The present work reports the synthesis of two types of complexes BITC:β-CD (IC-1) and BITC:hp-β-CD (IC-2) using Hielsher UP200St ultrasonic device. To prepare the complexes 0.044 mol of BITC was added drop wise to 0.044 mol β-CD (or hp-β-CD) with minimal solvent mixture (ethanol:water::2:8). The mixture was heated until 60ºC under continuous magnetic stirring. The solution was sonicated for 900 sec (3 sets of 300 secs each) at 180 W using Hielsher UP200St ultrasonic devices. The end products were obtained by lyophilisation. The method offers an efficient way to save time and energy consumption. 2.3. Characterization Techniques X-ray diffraction studies were conducted using Panalycital X’pert Pro. XRD with CuK radiation at a scanning speed of 10◦C/min. Field emission scanning electron microscopy and Energy dispersion X-ray spectroscopy analysis (FESEM-EDS) was carried out with HITACHI-SU8010 and BROOKER-XFLASH. SDT-Q-600 (TA instruments New Castle, DE) was used to investigate the thermal behaviour. The measurements were conducted using alumina pans, under nitrogen atmosphere with the flow rate 100.0 ml min-1. The samples were heated (~5 mg) at a constant heating rate 5°C min-1 to the desired temperature. The minimum energy optimization studies for the reactants and synthesized complexes were conducted using Gaussian 03 package with Gauss view 3.0. The structures were fully optimised using DFT-B3LYP method. FTIR spectra was recorded using Perkin-Elmer, Beaconsfield, Buck, U.K. spectrophotometer with a resolution of 4 cm−1 equipped with AgCl windows in the spectral region of 4000-500 cm-1. JASCO V 530 (4-21, Sennin-cho 2-chome, Hachioji, Tokyo 193-0835, Japan) model spectrophotometer was used to obtain UV-visible
absorption spectra with a precision of ± 0.2 nm using 1 cm path length quartz cell. CHNS-O analyser thermo scientific flash 2000 organic elemental analyser was employed for elemental analysis. The Phase solubility studies were conducted at three temperatures 25, 30 and 37˚C using excess amount of BITC and varying concentrations of β-CD/hp-β-CD (4-20 mmol L−1) using a reported method by Higuchi and Connors [12] with slight improvisation (S2,Supporting Information) 2.4. Confocal Experiment MDA-MB-231 breast cancer cells at a density of 2 × 104 were plated in 24 well tissue culture plate over sterile glass cover-slips and cultured overnight to adhere. Following day, spent culture media was removed and cells were exposed to BITC, IC-1 and IC-2 at 10 µg/ mL concentration of BITC prepared in cell culture media and incubated for 2 hr at standard cell culture conditions. After the incubation time, culture media containing treatments were removed and cells were washed with pre-warm phosphate buffered saline. Coverslips were mounted on cleaned degreased slides and visualized under inverted fluorescent microscope with 488 nm excitation and 500-550 nm emission wavelength (NIKON Eclipse). All images were acquired at 60X magnification. 2.5. Stability Studies The stability studies of free BITC and ICs were undertaken using 2mM of BITC and equivalent IC solutions using minimum amount of ethanol. Their comparative studies were observed at ambient temperature, using ultra violet exposure. Equal aliquots of solution were taken after a fixed interval of time and analysed with the help of UV-vis spectroscopy and the concentration of BITC was determined. The absorption graph was plotted where the percentage of remaining BITC and proportion of BITC degraded to its therapeutically dormant form in IC was taken on the ordinate axis and time interval on the abscissa. 2.6. Haemolysis Study The use of cyclodextrin based systems for nutraceutical delivery is subjected to meticulous interrogations to ascertain the nontoxicity of the formulation, a vital parameter for determining haemolytic toxicity. Whole blood was collected from healthy volunteers in heparinized centrifuge tube and red blood cells were pelleted by centrifugation at 2500 rpm for 10 min. After the removal of plasma, the cells were washed thrice with phosphatebuffered saline (PBS). Finally, haematocrit was to get 2% cell suspension and used for the assay. For haemolysis test, 1ml of the diluted haematocrit suspension in PBS was mixed with
1 ml of the test compound (BITC) with concentrations ranging from 2-1000 µg/ml as well as IC-1 and IC-2, containing equivalent BITC concentration. Then, these mixtures were incubated at 37 °C for 45 minutes. At the end of incubation, suspension was centrifuged and the optical density (OD) of supernatant was measured at 540 nm. 1% Triton-X 100 and PBS were used as positive and negative controls respectively. Percentage hemolysis was calculated by using the following formula: Haemolysis (%) = AS-AN/AP-AN
(1)
Where, AS, AP, and AN depicts the optical density values at 540 of sample, positive control, and negative control, respectively. 2.7. Mononuclear blood cells safety study The segregation of mononuclear cells (MNCs) from circulatory blood of healthy human volunteers was done by vene-puncture in heparinized vials. The protocol as elaborated by Fuss et al.[13] was followed with modifications. Briefly, blood was diluted with PBS in 1:1 ratio and was carefully layered on lymphocyte separation media (Hisep-LSM 1077 from Himedia, India) and centrifuged at 400×g for 30 min at 20 °C. After centrifugation, plasma and plasma containing supernatant was removed and the band of MNCs was cautiously aspirated to a fresh sterile tube. It was further washed with PBS and then centrifuged at 250×g 10 min to remove the trace of platelets. The step was repeated twice to obtain an almost pure population of MNCs.The viability was confirmed using trypan blue exclusion test in modified Neubauer’s improved chamber. MNCs were then suspended at 1 × 106 cells/ml in RPMI 1640 complete medium supplemented with 10% FBS, 1% Penicillin, Streptomycin and 0.25% Amphotericin B and 2 mM L-glutamine and seeded in U-bottom sterile 96 well plate. PBMCs were then treated with β-CD and hp-β-CD in similar amounts of 1-1000 µg-ml of BITC loaded IC-1 and IC-2 complexes and incubated in a 5% CO2 incubator at 37°C for 48 h. After 48 h completion, drug containing media was removed after centrifuging the plate at 250 ×g for 10 min and 10 % v/v MTT (5mg/ml) was added in the cultured cells followed by incubation at 37 °C for 4 h. After 4 h, MTT containing media was removed and formazan crystals were dissolved in 100 µL DMSO and optical density was measured at 570 nm. Untreated cells were used to serve as control and % viability was calculated by normalizing the optical density of treated cells against optical density of untreated control cells. 2.8. Antimicrobial Studies.
The antimicrobial activity of the free BITC and the two ICs were tested against Staphylococcus aureus ATCC 29213 by disk-diffusion assay. Briefly, 5 µg of the testing agents were loaded on to sterile 6-mm paper disks and the disks were allowed to dry. These disks were then placed on Mueller Hinton agar plate pre-inoculated with a bacterial suspension set to McFarland standard 0.5, so as to give a confluent lawn of growth after incubation. The plates were incubated for 24 h at 37°C. Diameters of the zones of the growth inhibition (ZOI) were measured up to the nearest whole millimetres. 3. Results and discussion 3.1. Embedding Ratio `
The amount of BITC entrapped in the ICs was determined spectrophotometrically.
The embedding ratios of BITC in β-CD and hp-β-CD i.e. IC-1 and IC-2 are 96.50% ± 0.62 and 95.01% ± 0.45, respectively. The high value of embedding ratios (96.5%) effectively suggested that the smaller molecular weight compound like BITC was suitable for inclusion in β-CD and modified β-CD. Only trace amounts of BITC were observed on the surface of the ICs formed by probe sonication method. These observations indicated that the probe sonication method provided good contact conditions between the guest (BITC) and host (β CD/hp-β-CD), thus ensuring higher complexation efficiency. The sample preparation for calculating the embedding ratios are given in S3 (Supporting Information) Li et al. [11] and Yuan et al. [14] have reported embedding ratio of 86.4% and 94.9% respectively for BITC in β-CD. But by using advanced probe sonication technique the embedding ratio gets further increased to 96.5% while the time required for fabrication was reduced to 15 min as compared to 3 hrs reported in literature [11,14]. Thus, from the available results it can, therefore, be safely deduced that sonication increased the emulsification degree of BITC as well as the contact area, which in turn improved the embedding ratio. It is the higher embedding ratio that provided feasibility for further application. 3.2. XRD XRD is an insightful method for the detection and characterization of CD complexes in powder or microcrystalline states [15-19]. Formation of ICs was confirmed [15-19] by the presence of diffuse diffraction patterns, appearance of new peaks or disappearance of characteristic peaks. Figure 1 shows the XRD patterns of β -CD, hp-β-CD and ICs.
Crystalline nature of β-CD was well evident in the XRD patterns depicted by intense and sharp peaks between 10 º and 60º (2θ 10.55, 12.44, 13.5, 18.2, 22.42 and 26.71) [17]. Moreover, this pattern was in conformity with the cage-type packing in the molecular arrangement of β –CDs [18]. The diffraction pattern of IC-1 showed the obvious variations on comparing with the obtained pattern of β-CD. The peaks at 2ϴ=10.55 disappeared and broader peaks at 12 and 18 appeared. These differences in the diffractogram of IC could be correlated and accounted for variations in the molecular arrangements of β-CD from cagetype to channel-type organization, which is an inviolable authentication of IC genesis [19].
Fig. 1 X-Ray powder diffraction diagrams of (a) β-CD (b) IC-1 (c) hp-β-CD (d) IC-2 Since BITC is liquid at room temperature, the corresponding XRD pattern could not be obtained. However, the literature [20,21] proclaims that the genesis of an IC between hpβ-CD and guest would not be a simple superposition of diffractrograms of the two compounds. The absence of any crystalline peak in the XRD pattern of hp-β-CD indicated its amorphous nature [20,21]. The obtained XRD of IC-2 showed an increase in intensity accompanied by change in the shape of the characteristic peak and appearance of two new peaks at 41.9 and 48.7o. It can be taken as an important evidence of the formation of IC-2. 3.3 Thermogravimetric Analysis The non-isothermal thermogravimetry (TG) with a continuous rise in temperature is one of the simplest and most frequently used procedure for studying the thermal behaviour of any formulation. TGA was bestowed to comprehend the thermal behaviour of synthesized
ICs. The values of activation energy; Ea, was calculated using several methods [22-26]. The TG curves of β-CD, hp-β-CD, IC-1 and IC-2 are depicted in Figure 2. BITC is a volatile compound and quickly loses mass from 80 oC and 165 oC.[11]. On correlating the decomposition curves of the reactants and the complexes formed, it was observed that while BITC decomposed in a single step (as reported in literature), β-CD, hp-β-CD, and the ICs decomposed following two step mechanism (Figure 2). The first step was the dehydration step in all the cases. The TG spectra of IC-1 and IC-2 were compared with their standard host moities viz., β-CD and hp-β-CD respectively. TG plots exhibited high thermal stability with distinct decomposition track. The higher decomposition temperature of ICs (360 ºC) relative to BITC was an indication of the former’s surpassing thermal stability. The first decomposition of ICs was observed between 80 oC to 120 oC which could be attributed to the decomposition of pure BITC (100 oC). Therefore, BITC engulfed in the cavity of the host molecules started to decompose in the same temperature range. Analogous decomposition results were also observed by Macedo et al.[27] and Li et al.[11].
Fig. 2 TG curves for (a) β-CD and IC-1 (c) hp-β-CD and IC-2 To determine the thermodynamic parameters, five empirical methods (Equations 2-9) based on single heating rate were used. Detailed calculations based on Coats–Redfern [22] (C), Madhusudanan Krishnan–Ninan [23] (M), Wanjun–Yuwen–Hen–Cunxin [24] (W), Van Krevelen [25] (K) and Horowitz–Metzger [26] (H), methods were reported in our previous work [28]. The integral function of conversion g(α) were surmised using parameters with all the five methods.(Figure 3)
variation
Fig. 3 Linear Fit plots obtained by (a) Coats–Redfern (C), (b) Madhusudanan–Krishnan– Ninan (M), (c) Wanjun–Yuwen–Hen–Cunxin (W), (d) van Krevelen (K) methods and (e) Horowitz–Metzger (H) methods. (1) In the (C) method,[22] the function g(α) is expressed as
g (α) =
ART 2 βE
2RT − E / RT 1 − E e
(2)
Applying ln the equation becomes:
− ln
The quantity ln
g (α) AR 2RT E = − ln 1− + 2 βE E RT T
(3)
AR 2RT 1− is constant for most values of E and over the temperature range βE E
in which most reactions occur. However, both E and A could vary with the experimental heating rate.
− log
− log
1 − (1 − α)1−n AR 2 RT E = log 1− − 2 βE E 2.303RT T (1 − n)
− log(1 − α) AR 2RT E = log 1− − 2 βE E 2.303RT T
for n ≠1
(4)
for n=1
(5)
(2) In the (M) method,[23] the equation used has the form:
− ln
g (α) AR E = − ln + 3.7678 − 1.9206 ln E − 0.12040 1.9206 βE RT T
(6)
where the symbols have their usual significance. (3) Wanjun–Yuwen–Hen–Cunxin [24] and Van Krevelen [25] used different approximations. (W) method [24]
− ln
g ( α) AR E = − ln + 3.6350 − 1.8946 ln E − 1.0014 1.8946 βE RT T
(7)
(4) (K) Method [25] Ea RTm A(0.368 / Tm ) ln g (α) = ln Ea + 1) β( RTm
(5)
+ E a + 1 ln T RT m
(8)
A new parameter ‘T=Tm+Ɵ’ was introduced by Horowitz–Metzger [26] (H). when the
reaction order was 1, the final expression became:
ln ln g (α) =
Eθ RTm2
(9)
The evaluated activation energy was found to be nearly the same for all methods. The apparent activation energy and thermal behaviour were evaluated as : EIC-2>EIC-1 (Table 1) indicating that IC-1 formed a more stable complex as compared to IC-2 and therefore, lower energy was required for decomposition. The TG analysis also gave an evidence of the stoichiometric ratio of formed complexes i.e. IC-1 and IC-2. Bai et al.[29] devised stoichiometric ratios of four guest molecules: 4-cresol, benzyl alcohol, ferrocene and decanoic acid with α-CD in solid-state using TG analysis accurately. Table 1: Activation energy and regression coefficient values obtained using different
methods. IC-1 E/kJ mol-1 C 10.782 M 10.630 W 10.912 K 8.917 H 9.049
R 0.996 0.998 0.999 0.998 0.997
IC-2 E/kJ mol-1 14.690 14.658 14.578 13.752 13.706
R 0.999 0.998 0.999 0.997 0.997
Stoichiometric ratio of BITC:host (β-CD/hp-β-CD) could be expressed as 1:n, according to the proposed method, if equation (10) is satisfied. n M guest – 2M H2O ≤
≤ n M guess
(10)
where Mguest, MH2O and MCD are the molar weight of BITC, water and host molecule respectively while Wpure CD, and W’pure CD depicted the mass of the total host moiety (β-CD /hp-β-CD) with and without BITC, respectively. Thus, substituting the values obtained from thermograms in equation 10 the ratio was estimated to be close to 1:1. Few researchers also elaborated the stoichiometry of the formed IC [30,31]. Bru et al.[31] reported that 1:1 IC had the highest correlation coefficient. The current study also affirmed the formation of 1:1 stoichiometric ratio for IC 3.4. Molecular Modelling (a)
(b)
Fig. 4 Minimum energy optimized structure for (a) IC-1 (b) IC-2
Molecular modelling calculations were carried out in order to further scrutinize the mechanism of formation IC between BITC and CDs (β-CD /hp-β-CD). Semi empirical quantum mechanical calculations using DFT-B3LYP programme were executed to evaluate the exact location of the guest (BITC) in the host cavity (β-CD /hp-β-CD). Using Gaussian software, the minimum energy for optimized structures of the complexes were obtained as: BITC (-761.88029315 a.u.), β-CD (-4249.44604236 a.u.), hp-β-CD (-5563.46323029 a.u.), IC-1(-5035.40905517 a.u.), IC-2(-6278.91533339 a.u.). The 3-D geometrical structures obtained by energy minimization are shown in Figure 4. It was well confirmed from the structures obtained that 1:1 complex was formed.
3.5. FESEM A qualitative method, SEM, was employed to visualise the surface morphological characteristics of IC. Bulani et al.[32] revealed that β-CD exhibited crystalline parallelogram structure while hp-β-CD displayed spherical crystals with cavities structures were reported by Zhang et al.[8]. For the prepared IC-1 and IC-2 by probe sonication method, the SEM pictures indicated flake like structures with irregular parallelogram shape. Significant changes in the crystalline habitus, modification in the shape of crystals and aspect is suggestive of the proof of the formation of ICs with CDs. The differences in the reported crystalline states of the raw materials and that of the product as seen under electron microscope indicated the formation of the IC [33]. Figures 5(c) and (d) depicted the typical EDS spectra for identifying the elemental composition of IC-1 and IC-2. It showed well defined peaks of carbon, oxygen, nitrogen and sulphur. No other peak corresponding to any impurity was seen in any of the spectra. This authenticated the purity of the formed complexes [8]. 333
The preliminary studies using FT-IR, UV-visible spectroscopy and elemental analysis
were also conducted for confirming the complex formation and estimating the purity of the product formed. The absorption peak at 2040 cm-1 which was characteristic of the vibration of N=C=S group in pure BITC was masked in IC, thus, confirming the formation of IC. Furthermore, the FTIR spectrum of IC-1 and IC-2 showed modification of signals associated with BITC and CDs. Comparison studies show that the spectra of IC-1 and IC-2 were not completely congruent with β-CD and hp-β-CD respectively. The bands obtained at 1088, 1183, 1325, 1544 and 1623cm-1 had also either shifted or diminished.UV-visible absorption
studies have also proved to be quite useful in exploring the structural changes and formation of ICs. It was observed that the absorption peaks of IC-1 and IC-2 were deformed and the absorbance value decreased as compared to native BITC which appeared as a single broad band at 254 nm. There was nearly no influence of each CD on the peak wavelength of BITC, however, the studies on ICs revealed a slightly shifted super-imposed peak at 254 nm indicating complex formation. CHNS-O analysis of β-CD, hp-β-CD and IC-1 and IC-2 are recorded in (Table S1). The observed % ages were found to be in close agreement with the calculated values supporting the formation of ICs. Detailed information has been given in (S4, Supporting Information)
(a)
(C)
(b)
(d)
Fig. 5 FESEM images of (a) IC-1 (b) IC-2; EDS images of (c) IC-1 (d) IC-2
The solubility of BITC in the aqueous media got enhanced with the increase in the concentrations of β-CD and hp-β-CD in the solution. The variations in the solubility of the bioactive agent was analysed to evaluate the stability constants and stoichiometric ratio of the obtained ICs. The phase solubility curves obtained at three temperatures (Figure S3, Supporting Information) indicated the linear increase in solubility with rise in temperature. The plot parameters suggested an AL type, indicating the formation of 1:1 IC. The slope of straight line was less than 1 for all the plots, thus giving an indirect proof of the formation of 1:1 complex [34]. The plots fortified the results obtained by thermal analysis and gaussian calculations. The stability constants (KC) for the ICs were calculated from the linear plots, (Figure S5 Supporting Information), considering 1:1 stoichiometry and the values obtained using Van’t Hoff plot have been listed in Table 2. The decrease in the value of stability constant with the increase in temperature indicated an exothermic process. The results were in comparison with the other reports reported in literature on the IC formation of BITC [35,36].
Table 2: Aqueous solubility of BITC in the absence of CDs (So) solubility, stability constants
(Kc) and thermodynamic parameters Temperature(ºC) So(mmol-1) (10-4) β-CD 25 1.17 30 1.37 37 1.40 hp-β-CD 25 9.20 30 12.10 37 18.10
Kc (M-1 ) -∆G -∆H -∆S (kJmol-1) (kJ mol-1) (J K-1mol-1) 391.69 264.13 151.40
12.44 14.05 15.39
14.40
75.77 67.55 61.19
278.60 164.80 115.70
12.11 12.85 13.95
12.50
88.50 85.20 83.32
The thermodynamic parameters were also calculated using the phase solubility data (equations (2) and (3)). The negative value of enthalpy change (-∆H) indicated the exothermic interaction between BITC and CDs (β-CD /hp-β-CD). The negative value of ∆G was indicative of the spontaneous nature of the interaction process. The restricted rotation of the encompassed molecule around the symmetry axis in the obtained complexes could be accounted for the negative value of entropy [36]. 3.6. Confocal Microscopy Overlay photomicrographs (Figure 6) were obtained using confocal laser scanning microscope. These images were acquired with the sole purpose of visualisation of cellular uptake of BITC alone, if it permeated the cellular membrane and further deciphering if the inclusion complexes hindered its intracellular localization. A specific wavelength for fluorescence signal detection was not reported in the literature. Specimens were scanned at various permutations and combinations of excitation and emissions possible on Nikon Eclipse. Of all the tested values of excitation emisions (combined) the most appropriate combinations was found to be
488 nm (excitation) and 500-550 nm (emission). With this
optical configuration, images were acquired at 60X Nikon Plan Apo objective with 1.40 numerical aperture.
Fig. 6 Confocal photomicrographs of (a) control (b) BITC (c) IC-1 (d) IC-2
Photomicrographs in Figure 6 showed that BITC was located inside the cytoplasm of the cells under all the conditions whether cells were treated with BITC alone or incorporated inside the inclusion complex IC-1 or IC-2 at more or less equal level as indicated by fluorescent intensity. BITC could easily migrate across the plasma membrane of cells and inclusion complexes posed no obstruction for cellular migration of active moiety. 4. Stability Studies
Despite having excellent anticancer properties, BITC is volatile, insoluble in water and unstable in its nascent form [37]. It is a light sensitive molecule and tended to degrade rapidly in UV light. The UV energy absorbed by BITC either dissipated as heat energy or lead to the formation of decomposition products. Therefore, the developed formulation was designed to protect the drug from UV degradation. An aliquot of 30 µM BITC solution in ethanol and prepared ICs in water were chosen for study. It was exposed to UV light at 254 nm (short range UV).
BITC IC-1 IC-2 3.0
Absorban ce (a.u.)
2.5 2.0 1.5 1.0 0.5 0.0
100 80
Ti 60 m e (m 40 in )
20 0
Fig. 7 UV Stability studies of BITC, β-CD and hp- β-CD
Figure 7 shows the content changes of active ingredient in BITC, IC-1 and IC-2 under the illumination of UV light. The experiment was carried out up to 120 minutes. The absorbance value of BITC in ethanol falls dramatically during the first 20 min. However, that of IC-1 and IC-2 decreased slightly during the same period. It was found that 75.9 % BITC in ethanol degraded in 26 min while even after the exposure to UV light for 120 min, the BITC in IC-1 and IC-2 retained its content (Figure. 7). Therefore, it was concluded that cyclodextrin acted as a protective unit and prevented the degradation of BITC. IC-1 degraded only 8.1% while IC-2 degraded 8.3% in 120 min. Accordingly, the UV stability of active ingredient (BITC) in IC’s was remarkably improved by comparison with raw BITC. The results, therefore, clearly indicated that IC with CDs might be a suitable medium to control the degradation of light sensitive molecules. When exposed to UV light, the CDs on the active ingredient acted as a protective barrier that resulted in the weakening of the degradation effect of ultraviolet radiations thus augmenting the stability. This reinforced stability was ascribed to the perfect encapsulation of BITC in the cavity of CDs by the ultrasonication methodology. 5. Haemolysis Study
Present haemolysis study investigated the use of β-CD and hp- β-CD as safe delivery vehicles for administration of BITC and for obtaining its maximum therapeutic benefits. To prove the nontoxicity of the formulation, haemolytic toxicity studies were conducted. In current haemolytic study free BITC, β-CD, hp-β-CD and BITC encapsulated cyclodextrins i.e. IC-1 and IC-2 were compared for extent of haemolysis. The haemolysis profile (Figure 8)
obtained for all concentrations of the formulation was found to be well under the tolerance limit (< 5%).This suggested that BITC could be safely administered using β-CD and hp-βCD, provided both are safe to other blood cellular components e.g. nucleated cells. 5.1. Mononuclear blood cells safety study Similar to haemolysis test, viability of mononuclear cells present in the blood after drug exposure was another prerequisite to prove the safety of any drug carriers. Both the carriers: β-CD and hp-β-CD were tested to check any cytotoxicity to nucleated components of blood including cells of lymphoid and myeloid lineage. Except for the highest concentration of 1000 µg/ml, none of the concentrations used for treatment showed significant toxicity and in all the treatment levels, viability was more than 95 percent. This nontoxic nature of both carriers validated the suitability of their use as delivery agents.
Fig. 8 (a) Haemolysis studies of BITC, β-CD, hp- β-CD, IC-1 and IC-2 (b) Viability of
mononuclear cells in presence of β-CD and hp- β-CD
6. Antimicrobial Studies
BITC is a promising antimicrobial [2,36,38] agent presently constrained by its low solubility and high volatility. Since the two ICs proposed in the present study helped in overcoming these limitations, various experiments were carried out in order to confirm that these ICs retained the antimicrobial properties of the free/bulk-phase drug. The mean diameter of the zones of growth inhibition obtained against S. aureus with free/bulk-phase drug, β-CD BITC IC and hp-β-CD BITC IC were 75±0, 54±6 and 56±1 mm respectively. Thus, a significant amount of antimicrobial activity was retained by the active drug in ICs, suggesting their excellent suitability as antimicrobial candidates.
7. Conclusions
The complexation behaviour of BITC with β-CD and hp-β-CD have been investigated by absorption spectroscopy, FTIR, TGA, FESEM, EDS, CHNS-O, XRD and molecular modelling. The results demonstrated that BITC was complexed with β-CD and hp-β-CD to form an IC by the ultra-sonication method in molar ratio 1:1. The results of trapping efficiency displayed IC as an enormously efficient system for complexation of the active component. The thermal stability of BITC was improved in complexes, as indicated by the elaborate thermogravimetric analysis. The results are presumed to be related to the association reactions occurring during embedding. The enhanced UV stability substantiated that β-CD and hp-β-CD were suitable hosts for low molecular weight and volatile isothiocyanate such as BITC. Further they acted as protective moieties and hence, suppressed degradation and reinforced its stability. A significant level of antimicrobial activity was retained by the active drug in both the as synthesized ICs, affirming them as phenomenal antimicrobial candidates.
Conflict of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Acknowledgements
KK would like to thank the DST for funding and INSPIRE faculty award. KK and SKM are thankful for financial assistance to DST-PURSE-II New Delhi, India
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FIGURES Fig. 1 X-Ray powder diffraction diagrams of (a) β-CD (b) IC-1 (c) hp-β-CD (d) IC-2 Fig. 2 TG curves for (a) β-CD and IC-1 (c) hp-β-CD and IC-2 Fig. 3 Linear Fit plots obtained by (a) Coats–Redfern (C), (b) Madhusudanan–Krishnan–
Ninan (M), (c) Wanjun–Yuwen–Hen–Cunxin (W), (d) van Krevelen (K) methods and (e) Horowitz–Metzger (H) methods. Fig. 4 Minimum energy optimized structure for (a) IC-1 (b) IC-2 Fig. 5 FESEM images of (a) IC-1 (b) IC-2; EDS images of (c) IC-1 (d) IC-2 Fig. 6 Confocal photomicrographs of (a) control (b) BITC (c) IC-1 (d) IC-2 Fig. 7 UV Stability studies of BITC, β-CD and hp- β-CD Fig. 8 (a) Haemolysis studies of BITC, β-CD, hp- β-CD, IC-1 and IC-2 (b) Viability of
mononuclear cells in presence of β-CD and hp- β-CD
Fig. 1 X-Ray powder diffraction diagrams of (a) β-CD (b) IC-1 (c) hp-β-CD (d) IC-2
Fig. 2 TG curves for (a) β-CD and IC-1 (c) hp-β-CD and IC-2
Fig. 3 Linear Fit plots obtained by (a) Coats–Redfern (C), (b) Madhusudanan–Krishnan–
Ninan (M), (c) Wanjun–Yuwen–Hen–Cunxin (W), (d) van Krevelen (K) methods and (e) Horowitz–Metzger (H) methods.
(a)
(b)
Fig. 4 Minimum energy optimized structure for (a) IC-1 (b) IC-2
(a)
(C)
(b)
(d)
Fig. 5 FESEM images of (a) IC-1 (b) IC-2; EDS images of (c) IC-1 (d) IC-2
Fig. 6 Confocal photomicrographs of (a) control (b) BITC (c) IC-1 (d) IC-2
BITC IC-1 IC-2 3.0
Absorba nce (a.u .)
2.5 2.0 1.5 1.0 0.5 0.0
100 80
Ti 60 m e (m 40 in 20 ) 0
Fig. 7 UV Stability studies of BITC, β-CD and hp- β-CD
Fig. 8 (a) Haemolysis studies of BITC, β-CD, hp- β-CD, IC-1 and IC-2 (b) Viability of
mononuclear cells in presence of β-CD and hp- β-CD
Highlights 1. Ultrasonication method : an efficient way to maximize the embedding ratios; Time reduction and energy efficient 2. Stability studies : Thermal and UV stability 3. 1:1 ratio complex confirmed through phase solubility studies, TGA and molecular modelling.
4. Haematological safety and cellular uptake data gives direction to in-depth analysis for its exploitation of anti-tumour activity.
S1350-4177(17)30164-5 http://dx.doi.org/10.1016/j.ultsonch.2017.04.007 ULTSON 3640
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
1 February 2017 4 April 2017 5 April 2017
Please cite this article as: S. Uppal, K. Kaur, R. Kumar, N.K. Kahlon, R. Singh, S.K. Mehta, Encompassment of Benzyl Isothiocyanate in Cyclodextrin using Ultrasonication Methodology to enhance its stability for biological applications, Ultrasonics Sonochemistry (2017), doi: http://dx.doi.org/10.1016/j.ultsonch.2017.04.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Encompassment of Benzyl Isothiocyanate in Cyclodextrin using Ultrasonication Methodology to enhance its stability for biological applications Shivani Uppala , Khushwinder Kaura*, Rajendra Kumarb, Nakshdeep Kaur Kahlonc, Rachna Singhc, S.K. Mehtaa* a
Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University, Chandigarh 160 014, India
b
UGC Centre of Excellence in Nanomaterials, Nanoparticles, Nanocomposites, Panjab University Chandigarh 160 014, India
c
Department of Microbial biotechnology, Panjab University Chandigarh 160 014, India
*Corresponding author: Khushwinder Kaur Tel: +919872040447; Fax: +91-172-2545074; Email: [email protected]
Abstract The use of methodical and innovative sonication method has been explored for the fabrication of inclusion complex of Benzyl Isothiocyanate, a potential anticancer and antimicrobial agent. The advancement involved investigation of inclusion behaviour, characterisation and an in-depth study of thermal and UV stability of Benzyl Isothiocyanate with cyclodextrins; β-CD and hp-β-CD. The sonication driven encompassment in cyclodextrins helped to overcome the hindrance of low solubility and high volatility. Investigations of physical and thermodynamic parameters using UV-visible spectroscopy, FTIR, XRD, TGA etc confirmed stability of inclusion complexes. Both β-CD and hp-β-CD based inclusion complexes retained the antimicrobial property of the free Benzyl Isothiocyanate, indicating their potential utility as antimicrobial agents. Haematological safety and cellular uptake data gives direction to in-depth analysis for its exploitation of antitumour activity.
Key words: cyclodextrin, benzyl isothiocyanate, thermal and UV stability, antimicrobial,
1. Introduction Benzyl isothiocyanate (BITC) is sulphur containing bioactive compound that exists predominantly in plants [1] like Tropaeolum mnajus L., Lepidium sativwm L., Carica papaya L. and Capparis flexuosa L. It shows excellent antibiotic activity against bacteria and fungi in vitro, possibly by targeting proteins and cell membrane integrity [2,3]. BITC induces cell cycle arrest, engender apoptosis and impede NFkB activation by triggering reactive oxygen species (ROS) [4,5]. The bioavailability of BITC in garden cress consumed by humans was approximately 14-50% due to the incapability of the sufficient therapeutic ingredient to be released from the vegetable by chewing so more than 50% of the delivered BITC is metabolized to its cysteine conjugate and ejected through urine [6]. Although BITC showed great potential preclinically but its effective impact during clinical investigation is impeded due to dearth of an adequate dosage form and poor bioavailability. β-Cyclodextrins (β-CD) and modified β-CDs are cyclic oligo glycosides having a polar, hydrophilic outside and a relatively nonpolar lipophilic inside [7]. The outer rim is hydrophilic, which is the reason behind the good solubility of CDs in aqueous media. Thus the solubility and stability of active guest-molecules gets amplified [8]. Low molecular weight compounds like BITC are best incorporated in β –CD [9]. Formation of inclusion complex (IC) of BITC is of enormous benefit in masking its acute odour, improving water solubility and meliorating its functional stability. In the present work, probe sonication, an extremely efficient, economical and time dipping technique capable of forming ICs [10] (reported in our previous work) has been used to form complexes of BITC with β-CD. Furthermore, complexation with modified β-CD and its detailed thermal studies have also been reported. The present report throws light on the enhanced stability of BITC in terms of thermodynamic parameters after complexation and its potential antimicrobial activity. This effortless, lucid and agile strategy not only produced the desirable outcomes quickly but also gave way to an environmental friendly methodology by avoiding the use of toxic solvents and saving endless labour. Further, both β-CD and modified β-CD (hp- β-CD) were investigated for haemato-compatibility as this interaction is expected after in vivo administration. Moreover, ICs loaded with BITC were able to permeate in vitro growing cancer cells equivalent to bare BITC. The cellular uptake behaviour was not affected by either of the ICs.
2. Experimental set-up and methodology 2.1. Materials Benzyl Isothiocyanate (> 99%), β-CD, (> 99%), hp-β-CD, (> 99%), Ethanol, (99%) and Hexane (> 99%) were procured from Sigma-Aldrich, India. MDA-MB-231 cells were used for cell uptake studies. Peripheral human blood from healthy volunteer was extracted to isolate the haematocrit and mononuclear cells for haematological studies. Triple distilled water was utilized to prepare samples. The structures of BITC and the two CDs used are reported in Scheme S1 (Supporting Information) 2.2. Preparation of Inclusion Complex Li et al. [11] reported the preparation of IC of BITC with β-CD by magnetic stirring followed by freeze drying ultrasonic homogenization for 3 h at 40ºC. The present work reports the synthesis of two types of complexes BITC:β-CD (IC-1) and BITC:hp-β-CD (IC-2) using Hielsher UP200St ultrasonic device. To prepare the complexes 0.044 mol of BITC was added drop wise to 0.044 mol β-CD (or hp-β-CD) with minimal solvent mixture (ethanol:water::2:8). The mixture was heated until 60ºC under continuous magnetic stirring. The solution was sonicated for 900 sec (3 sets of 300 secs each) at 180 W using Hielsher UP200St ultrasonic devices. The end products were obtained by lyophilisation. The method offers an efficient way to save time and energy consumption. 2.3. Characterization Techniques X-ray diffraction studies were conducted using Panalycital X’pert Pro. XRD with CuK radiation at a scanning speed of 10◦C/min. Field emission scanning electron microscopy and Energy dispersion X-ray spectroscopy analysis (FESEM-EDS) was carried out with HITACHI-SU8010 and BROOKER-XFLASH. SDT-Q-600 (TA instruments New Castle, DE) was used to investigate the thermal behaviour. The measurements were conducted using alumina pans, under nitrogen atmosphere with the flow rate 100.0 ml min-1. The samples were heated (~5 mg) at a constant heating rate 5°C min-1 to the desired temperature. The minimum energy optimization studies for the reactants and synthesized complexes were conducted using Gaussian 03 package with Gauss view 3.0. The structures were fully optimised using DFT-B3LYP method. FTIR spectra was recorded using Perkin-Elmer, Beaconsfield, Buck, U.K. spectrophotometer with a resolution of 4 cm−1 equipped with AgCl windows in the spectral region of 4000-500 cm-1. JASCO V 530 (4-21, Sennin-cho 2-chome, Hachioji, Tokyo 193-0835, Japan) model spectrophotometer was used to obtain UV-visible
absorption spectra with a precision of ± 0.2 nm using 1 cm path length quartz cell. CHNS-O analyser thermo scientific flash 2000 organic elemental analyser was employed for elemental analysis. The Phase solubility studies were conducted at three temperatures 25, 30 and 37˚C using excess amount of BITC and varying concentrations of β-CD/hp-β-CD (4-20 mmol L−1) using a reported method by Higuchi and Connors [12] with slight improvisation (S2,Supporting Information) 2.4. Confocal Experiment MDA-MB-231 breast cancer cells at a density of 2 × 104 were plated in 24 well tissue culture plate over sterile glass cover-slips and cultured overnight to adhere. Following day, spent culture media was removed and cells were exposed to BITC, IC-1 and IC-2 at 10 µg/ mL concentration of BITC prepared in cell culture media and incubated for 2 hr at standard cell culture conditions. After the incubation time, culture media containing treatments were removed and cells were washed with pre-warm phosphate buffered saline. Coverslips were mounted on cleaned degreased slides and visualized under inverted fluorescent microscope with 488 nm excitation and 500-550 nm emission wavelength (NIKON Eclipse). All images were acquired at 60X magnification. 2.5. Stability Studies The stability studies of free BITC and ICs were undertaken using 2mM of BITC and equivalent IC solutions using minimum amount of ethanol. Their comparative studies were observed at ambient temperature, using ultra violet exposure. Equal aliquots of solution were taken after a fixed interval of time and analysed with the help of UV-vis spectroscopy and the concentration of BITC was determined. The absorption graph was plotted where the percentage of remaining BITC and proportion of BITC degraded to its therapeutically dormant form in IC was taken on the ordinate axis and time interval on the abscissa. 2.6. Haemolysis Study The use of cyclodextrin based systems for nutraceutical delivery is subjected to meticulous interrogations to ascertain the nontoxicity of the formulation, a vital parameter for determining haemolytic toxicity. Whole blood was collected from healthy volunteers in heparinized centrifuge tube and red blood cells were pelleted by centrifugation at 2500 rpm for 10 min. After the removal of plasma, the cells were washed thrice with phosphatebuffered saline (PBS). Finally, haematocrit was to get 2% cell suspension and used for the assay. For haemolysis test, 1ml of the diluted haematocrit suspension in PBS was mixed with
1 ml of the test compound (BITC) with concentrations ranging from 2-1000 µg/ml as well as IC-1 and IC-2, containing equivalent BITC concentration. Then, these mixtures were incubated at 37 °C for 45 minutes. At the end of incubation, suspension was centrifuged and the optical density (OD) of supernatant was measured at 540 nm. 1% Triton-X 100 and PBS were used as positive and negative controls respectively. Percentage hemolysis was calculated by using the following formula: Haemolysis (%) = AS-AN/AP-AN
(1)
Where, AS, AP, and AN depicts the optical density values at 540 of sample, positive control, and negative control, respectively. 2.7. Mononuclear blood cells safety study The segregation of mononuclear cells (MNCs) from circulatory blood of healthy human volunteers was done by vene-puncture in heparinized vials. The protocol as elaborated by Fuss et al.[13] was followed with modifications. Briefly, blood was diluted with PBS in 1:1 ratio and was carefully layered on lymphocyte separation media (Hisep-LSM 1077 from Himedia, India) and centrifuged at 400×g for 30 min at 20 °C. After centrifugation, plasma and plasma containing supernatant was removed and the band of MNCs was cautiously aspirated to a fresh sterile tube. It was further washed with PBS and then centrifuged at 250×g 10 min to remove the trace of platelets. The step was repeated twice to obtain an almost pure population of MNCs.The viability was confirmed using trypan blue exclusion test in modified Neubauer’s improved chamber. MNCs were then suspended at 1 × 106 cells/ml in RPMI 1640 complete medium supplemented with 10% FBS, 1% Penicillin, Streptomycin and 0.25% Amphotericin B and 2 mM L-glutamine and seeded in U-bottom sterile 96 well plate. PBMCs were then treated with β-CD and hp-β-CD in similar amounts of 1-1000 µg-ml of BITC loaded IC-1 and IC-2 complexes and incubated in a 5% CO2 incubator at 37°C for 48 h. After 48 h completion, drug containing media was removed after centrifuging the plate at 250 ×g for 10 min and 10 % v/v MTT (5mg/ml) was added in the cultured cells followed by incubation at 37 °C for 4 h. After 4 h, MTT containing media was removed and formazan crystals were dissolved in 100 µL DMSO and optical density was measured at 570 nm. Untreated cells were used to serve as control and % viability was calculated by normalizing the optical density of treated cells against optical density of untreated control cells. 2.8. Antimicrobial Studies.
The antimicrobial activity of the free BITC and the two ICs were tested against Staphylococcus aureus ATCC 29213 by disk-diffusion assay. Briefly, 5 µg of the testing agents were loaded on to sterile 6-mm paper disks and the disks were allowed to dry. These disks were then placed on Mueller Hinton agar plate pre-inoculated with a bacterial suspension set to McFarland standard 0.5, so as to give a confluent lawn of growth after incubation. The plates were incubated for 24 h at 37°C. Diameters of the zones of the growth inhibition (ZOI) were measured up to the nearest whole millimetres. 3. Results and discussion 3.1. Embedding Ratio `
The amount of BITC entrapped in the ICs was determined spectrophotometrically.
The embedding ratios of BITC in β-CD and hp-β-CD i.e. IC-1 and IC-2 are 96.50% ± 0.62 and 95.01% ± 0.45, respectively. The high value of embedding ratios (96.5%) effectively suggested that the smaller molecular weight compound like BITC was suitable for inclusion in β-CD and modified β-CD. Only trace amounts of BITC were observed on the surface of the ICs formed by probe sonication method. These observations indicated that the probe sonication method provided good contact conditions between the guest (BITC) and host (β CD/hp-β-CD), thus ensuring higher complexation efficiency. The sample preparation for calculating the embedding ratios are given in S3 (Supporting Information) Li et al. [11] and Yuan et al. [14] have reported embedding ratio of 86.4% and 94.9% respectively for BITC in β-CD. But by using advanced probe sonication technique the embedding ratio gets further increased to 96.5% while the time required for fabrication was reduced to 15 min as compared to 3 hrs reported in literature [11,14]. Thus, from the available results it can, therefore, be safely deduced that sonication increased the emulsification degree of BITC as well as the contact area, which in turn improved the embedding ratio. It is the higher embedding ratio that provided feasibility for further application. 3.2. XRD XRD is an insightful method for the detection and characterization of CD complexes in powder or microcrystalline states [15-19]. Formation of ICs was confirmed [15-19] by the presence of diffuse diffraction patterns, appearance of new peaks or disappearance of characteristic peaks. Figure 1 shows the XRD patterns of β -CD, hp-β-CD and ICs.
Crystalline nature of β-CD was well evident in the XRD patterns depicted by intense and sharp peaks between 10 º and 60º (2θ 10.55, 12.44, 13.5, 18.2, 22.42 and 26.71) [17]. Moreover, this pattern was in conformity with the cage-type packing in the molecular arrangement of β –CDs [18]. The diffraction pattern of IC-1 showed the obvious variations on comparing with the obtained pattern of β-CD. The peaks at 2ϴ=10.55 disappeared and broader peaks at 12 and 18 appeared. These differences in the diffractogram of IC could be correlated and accounted for variations in the molecular arrangements of β-CD from cagetype to channel-type organization, which is an inviolable authentication of IC genesis [19].
Fig. 1 X-Ray powder diffraction diagrams of (a) β-CD (b) IC-1 (c) hp-β-CD (d) IC-2 Since BITC is liquid at room temperature, the corresponding XRD pattern could not be obtained. However, the literature [20,21] proclaims that the genesis of an IC between hpβ-CD and guest would not be a simple superposition of diffractrograms of the two compounds. The absence of any crystalline peak in the XRD pattern of hp-β-CD indicated its amorphous nature [20,21]. The obtained XRD of IC-2 showed an increase in intensity accompanied by change in the shape of the characteristic peak and appearance of two new peaks at 41.9 and 48.7o. It can be taken as an important evidence of the formation of IC-2. 3.3 Thermogravimetric Analysis The non-isothermal thermogravimetry (TG) with a continuous rise in temperature is one of the simplest and most frequently used procedure for studying the thermal behaviour of any formulation. TGA was bestowed to comprehend the thermal behaviour of synthesized
ICs. The values of activation energy; Ea, was calculated using several methods [22-26]. The TG curves of β-CD, hp-β-CD, IC-1 and IC-2 are depicted in Figure 2. BITC is a volatile compound and quickly loses mass from 80 oC and 165 oC.[11]. On correlating the decomposition curves of the reactants and the complexes formed, it was observed that while BITC decomposed in a single step (as reported in literature), β-CD, hp-β-CD, and the ICs decomposed following two step mechanism (Figure 2). The first step was the dehydration step in all the cases. The TG spectra of IC-1 and IC-2 were compared with their standard host moities viz., β-CD and hp-β-CD respectively. TG plots exhibited high thermal stability with distinct decomposition track. The higher decomposition temperature of ICs (360 ºC) relative to BITC was an indication of the former’s surpassing thermal stability. The first decomposition of ICs was observed between 80 oC to 120 oC which could be attributed to the decomposition of pure BITC (100 oC). Therefore, BITC engulfed in the cavity of the host molecules started to decompose in the same temperature range. Analogous decomposition results were also observed by Macedo et al.[27] and Li et al.[11].
Fig. 2 TG curves for (a) β-CD and IC-1 (c) hp-β-CD and IC-2 To determine the thermodynamic parameters, five empirical methods (Equations 2-9) based on single heating rate were used. Detailed calculations based on Coats–Redfern [22] (C), Madhusudanan Krishnan–Ninan [23] (M), Wanjun–Yuwen–Hen–Cunxin [24] (W), Van Krevelen [25] (K) and Horowitz–Metzger [26] (H), methods were reported in our previous work [28]. The integral function of conversion g(α) were surmised using parameters with all the five methods.(Figure 3)
variation
Fig. 3 Linear Fit plots obtained by (a) Coats–Redfern (C), (b) Madhusudanan–Krishnan– Ninan (M), (c) Wanjun–Yuwen–Hen–Cunxin (W), (d) van Krevelen (K) methods and (e) Horowitz–Metzger (H) methods. (1) In the (C) method,[22] the function g(α) is expressed as
g (α) =
ART 2 βE
2RT − E / RT 1 − E e
(2)
Applying ln the equation becomes:
− ln
The quantity ln
g (α) AR 2RT E = − ln 1− + 2 βE E RT T
(3)
AR 2RT 1− is constant for most values of E and over the temperature range βE E
in which most reactions occur. However, both E and A could vary with the experimental heating rate.
− log
− log
1 − (1 − α)1−n AR 2 RT E = log 1− − 2 βE E 2.303RT T (1 − n)
− log(1 − α) AR 2RT E = log 1− − 2 βE E 2.303RT T
for n ≠1
(4)
for n=1
(5)
(2) In the (M) method,[23] the equation used has the form:
− ln
g (α) AR E = − ln + 3.7678 − 1.9206 ln E − 0.12040 1.9206 βE RT T
(6)
where the symbols have their usual significance. (3) Wanjun–Yuwen–Hen–Cunxin [24] and Van Krevelen [25] used different approximations. (W) method [24]
− ln
g ( α) AR E = − ln + 3.6350 − 1.8946 ln E − 1.0014 1.8946 βE RT T
(7)
(4) (K) Method [25] Ea RTm A(0.368 / Tm ) ln g (α) = ln Ea + 1) β( RTm
(5)
+ E a + 1 ln T RT m
(8)
A new parameter ‘T=Tm+Ɵ’ was introduced by Horowitz–Metzger [26] (H). when the
reaction order was 1, the final expression became:
ln ln g (α) =
Eθ RTm2
(9)
The evaluated activation energy was found to be nearly the same for all methods. The apparent activation energy and thermal behaviour were evaluated as : EIC-2>EIC-1 (Table 1) indicating that IC-1 formed a more stable complex as compared to IC-2 and therefore, lower energy was required for decomposition. The TG analysis also gave an evidence of the stoichiometric ratio of formed complexes i.e. IC-1 and IC-2. Bai et al.[29] devised stoichiometric ratios of four guest molecules: 4-cresol, benzyl alcohol, ferrocene and decanoic acid with α-CD in solid-state using TG analysis accurately. Table 1: Activation energy and regression coefficient values obtained using different
methods. IC-1 E/kJ mol-1 C 10.782 M 10.630 W 10.912 K 8.917 H 9.049
R 0.996 0.998 0.999 0.998 0.997
IC-2 E/kJ mol-1 14.690 14.658 14.578 13.752 13.706
R 0.999 0.998 0.999 0.997 0.997
Stoichiometric ratio of BITC:host (β-CD/hp-β-CD) could be expressed as 1:n, according to the proposed method, if equation (10) is satisfied. n M guest – 2M H2O ≤
≤ n M guess
(10)
where Mguest, MH2O and MCD are the molar weight of BITC, water and host molecule respectively while Wpure CD, and W’pure CD depicted the mass of the total host moiety (β-CD /hp-β-CD) with and without BITC, respectively. Thus, substituting the values obtained from thermograms in equation 10 the ratio was estimated to be close to 1:1. Few researchers also elaborated the stoichiometry of the formed IC [30,31]. Bru et al.[31] reported that 1:1 IC had the highest correlation coefficient. The current study also affirmed the formation of 1:1 stoichiometric ratio for IC 3.4. Molecular Modelling (a)
(b)
Fig. 4 Minimum energy optimized structure for (a) IC-1 (b) IC-2
Molecular modelling calculations were carried out in order to further scrutinize the mechanism of formation IC between BITC and CDs (β-CD /hp-β-CD). Semi empirical quantum mechanical calculations using DFT-B3LYP programme were executed to evaluate the exact location of the guest (BITC) in the host cavity (β-CD /hp-β-CD). Using Gaussian software, the minimum energy for optimized structures of the complexes were obtained as: BITC (-761.88029315 a.u.), β-CD (-4249.44604236 a.u.), hp-β-CD (-5563.46323029 a.u.), IC-1(-5035.40905517 a.u.), IC-2(-6278.91533339 a.u.). The 3-D geometrical structures obtained by energy minimization are shown in Figure 4. It was well confirmed from the structures obtained that 1:1 complex was formed.
3.5. FESEM A qualitative method, SEM, was employed to visualise the surface morphological characteristics of IC. Bulani et al.[32] revealed that β-CD exhibited crystalline parallelogram structure while hp-β-CD displayed spherical crystals with cavities structures were reported by Zhang et al.[8]. For the prepared IC-1 and IC-2 by probe sonication method, the SEM pictures indicated flake like structures with irregular parallelogram shape. Significant changes in the crystalline habitus, modification in the shape of crystals and aspect is suggestive of the proof of the formation of ICs with CDs. The differences in the reported crystalline states of the raw materials and that of the product as seen under electron microscope indicated the formation of the IC [33]. Figures 5(c) and (d) depicted the typical EDS spectra for identifying the elemental composition of IC-1 and IC-2. It showed well defined peaks of carbon, oxygen, nitrogen and sulphur. No other peak corresponding to any impurity was seen in any of the spectra. This authenticated the purity of the formed complexes [8]. 333
The preliminary studies using FT-IR, UV-visible spectroscopy and elemental analysis
were also conducted for confirming the complex formation and estimating the purity of the product formed. The absorption peak at 2040 cm-1 which was characteristic of the vibration of N=C=S group in pure BITC was masked in IC, thus, confirming the formation of IC. Furthermore, the FTIR spectrum of IC-1 and IC-2 showed modification of signals associated with BITC and CDs. Comparison studies show that the spectra of IC-1 and IC-2 were not completely congruent with β-CD and hp-β-CD respectively. The bands obtained at 1088, 1183, 1325, 1544 and 1623cm-1 had also either shifted or diminished.UV-visible absorption
studies have also proved to be quite useful in exploring the structural changes and formation of ICs. It was observed that the absorption peaks of IC-1 and IC-2 were deformed and the absorbance value decreased as compared to native BITC which appeared as a single broad band at 254 nm. There was nearly no influence of each CD on the peak wavelength of BITC, however, the studies on ICs revealed a slightly shifted super-imposed peak at 254 nm indicating complex formation. CHNS-O analysis of β-CD, hp-β-CD and IC-1 and IC-2 are recorded in (Table S1). The observed % ages were found to be in close agreement with the calculated values supporting the formation of ICs. Detailed information has been given in (S4, Supporting Information)
(a)
(C)
(b)
(d)
Fig. 5 FESEM images of (a) IC-1 (b) IC-2; EDS images of (c) IC-1 (d) IC-2
The solubility of BITC in the aqueous media got enhanced with the increase in the concentrations of β-CD and hp-β-CD in the solution. The variations in the solubility of the bioactive agent was analysed to evaluate the stability constants and stoichiometric ratio of the obtained ICs. The phase solubility curves obtained at three temperatures (Figure S3, Supporting Information) indicated the linear increase in solubility with rise in temperature. The plot parameters suggested an AL type, indicating the formation of 1:1 IC. The slope of straight line was less than 1 for all the plots, thus giving an indirect proof of the formation of 1:1 complex [34]. The plots fortified the results obtained by thermal analysis and gaussian calculations. The stability constants (KC) for the ICs were calculated from the linear plots, (Figure S5 Supporting Information), considering 1:1 stoichiometry and the values obtained using Van’t Hoff plot have been listed in Table 2. The decrease in the value of stability constant with the increase in temperature indicated an exothermic process. The results were in comparison with the other reports reported in literature on the IC formation of BITC [35,36].
Table 2: Aqueous solubility of BITC in the absence of CDs (So) solubility, stability constants
(Kc) and thermodynamic parameters Temperature(ºC) So(mmol-1) (10-4) β-CD 25 1.17 30 1.37 37 1.40 hp-β-CD 25 9.20 30 12.10 37 18.10
Kc (M-1 ) -∆G -∆H -∆S (kJmol-1) (kJ mol-1) (J K-1mol-1) 391.69 264.13 151.40
12.44 14.05 15.39
14.40
75.77 67.55 61.19
278.60 164.80 115.70
12.11 12.85 13.95
12.50
88.50 85.20 83.32
The thermodynamic parameters were also calculated using the phase solubility data (equations (2) and (3)). The negative value of enthalpy change (-∆H) indicated the exothermic interaction between BITC and CDs (β-CD /hp-β-CD). The negative value of ∆G was indicative of the spontaneous nature of the interaction process. The restricted rotation of the encompassed molecule around the symmetry axis in the obtained complexes could be accounted for the negative value of entropy [36]. 3.6. Confocal Microscopy Overlay photomicrographs (Figure 6) were obtained using confocal laser scanning microscope. These images were acquired with the sole purpose of visualisation of cellular uptake of BITC alone, if it permeated the cellular membrane and further deciphering if the inclusion complexes hindered its intracellular localization. A specific wavelength for fluorescence signal detection was not reported in the literature. Specimens were scanned at various permutations and combinations of excitation and emissions possible on Nikon Eclipse. Of all the tested values of excitation emisions (combined) the most appropriate combinations was found to be
488 nm (excitation) and 500-550 nm (emission). With this
optical configuration, images were acquired at 60X Nikon Plan Apo objective with 1.40 numerical aperture.
Fig. 6 Confocal photomicrographs of (a) control (b) BITC (c) IC-1 (d) IC-2
Photomicrographs in Figure 6 showed that BITC was located inside the cytoplasm of the cells under all the conditions whether cells were treated with BITC alone or incorporated inside the inclusion complex IC-1 or IC-2 at more or less equal level as indicated by fluorescent intensity. BITC could easily migrate across the plasma membrane of cells and inclusion complexes posed no obstruction for cellular migration of active moiety. 4. Stability Studies
Despite having excellent anticancer properties, BITC is volatile, insoluble in water and unstable in its nascent form [37]. It is a light sensitive molecule and tended to degrade rapidly in UV light. The UV energy absorbed by BITC either dissipated as heat energy or lead to the formation of decomposition products. Therefore, the developed formulation was designed to protect the drug from UV degradation. An aliquot of 30 µM BITC solution in ethanol and prepared ICs in water were chosen for study. It was exposed to UV light at 254 nm (short range UV).
BITC IC-1 IC-2 3.0
Absorban ce (a.u.)
2.5 2.0 1.5 1.0 0.5 0.0
100 80
Ti 60 m e (m 40 in )
20 0
Fig. 7 UV Stability studies of BITC, β-CD and hp- β-CD
Figure 7 shows the content changes of active ingredient in BITC, IC-1 and IC-2 under the illumination of UV light. The experiment was carried out up to 120 minutes. The absorbance value of BITC in ethanol falls dramatically during the first 20 min. However, that of IC-1 and IC-2 decreased slightly during the same period. It was found that 75.9 % BITC in ethanol degraded in 26 min while even after the exposure to UV light for 120 min, the BITC in IC-1 and IC-2 retained its content (Figure. 7). Therefore, it was concluded that cyclodextrin acted as a protective unit and prevented the degradation of BITC. IC-1 degraded only 8.1% while IC-2 degraded 8.3% in 120 min. Accordingly, the UV stability of active ingredient (BITC) in IC’s was remarkably improved by comparison with raw BITC. The results, therefore, clearly indicated that IC with CDs might be a suitable medium to control the degradation of light sensitive molecules. When exposed to UV light, the CDs on the active ingredient acted as a protective barrier that resulted in the weakening of the degradation effect of ultraviolet radiations thus augmenting the stability. This reinforced stability was ascribed to the perfect encapsulation of BITC in the cavity of CDs by the ultrasonication methodology. 5. Haemolysis Study
Present haemolysis study investigated the use of β-CD and hp- β-CD as safe delivery vehicles for administration of BITC and for obtaining its maximum therapeutic benefits. To prove the nontoxicity of the formulation, haemolytic toxicity studies were conducted. In current haemolytic study free BITC, β-CD, hp-β-CD and BITC encapsulated cyclodextrins i.e. IC-1 and IC-2 were compared for extent of haemolysis. The haemolysis profile (Figure 8)
obtained for all concentrations of the formulation was found to be well under the tolerance limit (< 5%).This suggested that BITC could be safely administered using β-CD and hp-βCD, provided both are safe to other blood cellular components e.g. nucleated cells. 5.1. Mononuclear blood cells safety study Similar to haemolysis test, viability of mononuclear cells present in the blood after drug exposure was another prerequisite to prove the safety of any drug carriers. Both the carriers: β-CD and hp-β-CD were tested to check any cytotoxicity to nucleated components of blood including cells of lymphoid and myeloid lineage. Except for the highest concentration of 1000 µg/ml, none of the concentrations used for treatment showed significant toxicity and in all the treatment levels, viability was more than 95 percent. This nontoxic nature of both carriers validated the suitability of their use as delivery agents.
Fig. 8 (a) Haemolysis studies of BITC, β-CD, hp- β-CD, IC-1 and IC-2 (b) Viability of
mononuclear cells in presence of β-CD and hp- β-CD
6. Antimicrobial Studies
BITC is a promising antimicrobial [2,36,38] agent presently constrained by its low solubility and high volatility. Since the two ICs proposed in the present study helped in overcoming these limitations, various experiments were carried out in order to confirm that these ICs retained the antimicrobial properties of the free/bulk-phase drug. The mean diameter of the zones of growth inhibition obtained against S. aureus with free/bulk-phase drug, β-CD BITC IC and hp-β-CD BITC IC were 75±0, 54±6 and 56±1 mm respectively. Thus, a significant amount of antimicrobial activity was retained by the active drug in ICs, suggesting their excellent suitability as antimicrobial candidates.
7. Conclusions
The complexation behaviour of BITC with β-CD and hp-β-CD have been investigated by absorption spectroscopy, FTIR, TGA, FESEM, EDS, CHNS-O, XRD and molecular modelling. The results demonstrated that BITC was complexed with β-CD and hp-β-CD to form an IC by the ultra-sonication method in molar ratio 1:1. The results of trapping efficiency displayed IC as an enormously efficient system for complexation of the active component. The thermal stability of BITC was improved in complexes, as indicated by the elaborate thermogravimetric analysis. The results are presumed to be related to the association reactions occurring during embedding. The enhanced UV stability substantiated that β-CD and hp-β-CD were suitable hosts for low molecular weight and volatile isothiocyanate such as BITC. Further they acted as protective moieties and hence, suppressed degradation and reinforced its stability. A significant level of antimicrobial activity was retained by the active drug in both the as synthesized ICs, affirming them as phenomenal antimicrobial candidates.
Conflict of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Acknowledgements
KK would like to thank the DST for funding and INSPIRE faculty award. KK and SKM are thankful for financial assistance to DST-PURSE-II New Delhi, India
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FIGURES Fig. 1 X-Ray powder diffraction diagrams of (a) β-CD (b) IC-1 (c) hp-β-CD (d) IC-2 Fig. 2 TG curves for (a) β-CD and IC-1 (c) hp-β-CD and IC-2 Fig. 3 Linear Fit plots obtained by (a) Coats–Redfern (C), (b) Madhusudanan–Krishnan–
Ninan (M), (c) Wanjun–Yuwen–Hen–Cunxin (W), (d) van Krevelen (K) methods and (e) Horowitz–Metzger (H) methods. Fig. 4 Minimum energy optimized structure for (a) IC-1 (b) IC-2 Fig. 5 FESEM images of (a) IC-1 (b) IC-2; EDS images of (c) IC-1 (d) IC-2 Fig. 6 Confocal photomicrographs of (a) control (b) BITC (c) IC-1 (d) IC-2 Fig. 7 UV Stability studies of BITC, β-CD and hp- β-CD Fig. 8 (a) Haemolysis studies of BITC, β-CD, hp- β-CD, IC-1 and IC-2 (b) Viability of
mononuclear cells in presence of β-CD and hp- β-CD
Fig. 1 X-Ray powder diffraction diagrams of (a) β-CD (b) IC-1 (c) hp-β-CD (d) IC-2
Fig. 2 TG curves for (a) β-CD and IC-1 (c) hp-β-CD and IC-2
Fig. 3 Linear Fit plots obtained by (a) Coats–Redfern (C), (b) Madhusudanan–Krishnan–
Ninan (M), (c) Wanjun–Yuwen–Hen–Cunxin (W), (d) van Krevelen (K) methods and (e) Horowitz–Metzger (H) methods.
(a)
(b)
Fig. 4 Minimum energy optimized structure for (a) IC-1 (b) IC-2
(a)
(C)
(b)
(d)
Fig. 5 FESEM images of (a) IC-1 (b) IC-2; EDS images of (c) IC-1 (d) IC-2
Fig. 6 Confocal photomicrographs of (a) control (b) BITC (c) IC-1 (d) IC-2
BITC IC-1 IC-2 3.0
Absorba nce (a.u .)
2.5 2.0 1.5 1.0 0.5 0.0
100 80
Ti 60 m e (m 40 in 20 ) 0
Fig. 7 UV Stability studies of BITC, β-CD and hp- β-CD
Fig. 8 (a) Haemolysis studies of BITC, β-CD, hp- β-CD, IC-1 and IC-2 (b) Viability of
mononuclear cells in presence of β-CD and hp- β-CD
Highlights 1. Ultrasonication method : an efficient way to maximize the embedding ratios; Time reduction and energy efficient 2. Stability studies : Thermal and UV stability 3. 1:1 ratio complex confirmed through phase solubility studies, TGA and molecular modelling.
4. Haematological safety and cellular uptake data gives direction to in-depth analysis for its exploitation of anti-tumour activity.