Inclusion complexation of sulfapyridine with α- and β-cyclodextrins: Spectral and molecular modeling study

Journal of Molecular Structure 1054–1055 (2013) 215–222

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Inclusion complexation of sulfapyridine with a- and b-cyclodextrins: Spectral and molecular modeling study N. Rajendiran ⇑, S. Siva, J. Saravanan Department of Chemistry, Annamalai University, Annamalai Nagar 608 002, Tamilnadu, India

h i g h l i g h t s  Inclusion complexes of SFP with

a- and b-cyclodextrin were investigated. a-CD and b-CD at pH  6.5.

 SFP drug formed 1:1 complex with

1

 Complex formation was confirmed by FTIR, DSC, SEM, H NMR, XRD and PM3 methods.  Part of pyridine ring of SFP is present inside the CD nanocavity.  Nanoparticles were observed from drug–CD complex in the presence of water by TEM.

a r t i c l e

i n f o

Article history: Received 11 August 2013 Received in revised form 23 September 2013 Accepted 23 September 2013 Available online 29 September 2013 Keywords: Sulfapyridine Cyclodextrins Inclusion complexation Nanoparticles Molecular modeling

a b s t r a c t The inclusion complexes of sulfapyridine (SFP) with a-CD and b-CD were investigated by absorption, fluorescence, time-resolved fluorescence, FTIR, DSC, XRD, 1H NMR, SEM, TEM and molecular modeling methods. The normal fluorescence takes place from locally excited (LE) state while twisted intramolecular charge transfer (TICT) is responsible for highly Stokes shifted fluorescence. The enhancement of TICT emission in both CDs suggesting that the inclusion process plays the major role in this emission. The spectral shifts revealed that part of pyridine ring of SFP is entrapped in the CDs cavities. TEM images confirmed round shaped nanoparticles with the average size about 20–50 nm were observed in SFP with aCD and b-CD inclusion complexes. PM3 calculations have suggested that the large stabilization of excited singlet state of SFP with twisted conformation occurring at the amide SAN bond between the electron donor group (aniline ring) and the electron acceptor group (pyridine ring). Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Cyclodextrins CDs belongs to the family of cyclic oligosaccharides and have been studied extensively as a host molecule in supramolecular chemistry [1–7]. CDs have a toroidal or cone shape with all the secondary hydroxyl groups located on the wider rim, while all the primary hydroxyl groups on the narrower rim (Fig. 1). The primary and secondary hydroxyls on the outside of the CDs makes it water-soluble. By good features of their shape and hydrophobic nature of cavity, CDs accommodate a variety of hydrophobic molecules, or part of it, inside their cavity through non-covalent interactions to form inclusion complexes [2]. In addition to this hydrophobic interaction, other interactions such as van der Waals forces, electrostatic interactions and hydrogen bonding interactions can also be driving forces for complex formation of guest molecules with CDs [3]. This ability has been widely used ⇑ Corresponding author. Tel.: +91 94866 28800; fax: +91 4144 238080. E-mail address: [email protected] (N. Rajendiran). 0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molstruc.2013.09.035

for analytical purposes [3,4]. The pharmaceutical value of CD is that they forming inclusion complexes with the hydrophobic drugs can increase aqueous solubility, enhance stability [5], decrease toxicity and effect of drugs, control drugs releasing and improve bioavailability [6,7]. Sulfonamides, a series of drugs containing the chemical structure of sulfanillic amide, are one of the most widely administered groups of antibiotics in human and animal husbandry as preventive and therapeutic agents for certain infections caused by gram-positive and gram-negative microorganisms, fungi and certain protozoa as well as their relatively low price [8–10]. Sulfapyridine (4-amino-N-pyridin-2-ylbenzenesulfonamide, SFP) (Fig. 1), is one of the widely used synthetic anti-microbial agent of the sulfonamide class having the bioactive p-amino group and is employed almost exclusively for the treatment of pneumonia. Recently, we investigated a series of sulfonamide derivatives with b-CD through experimental [11,12] and theoretical methods [13]. The study in aqueous solution demonstrated that the presence of intramolecular charge transfer (ICT) or twisted

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c

e

O

f N b

N

O

d g

S

Ha

4.2553 Å

216

d

(a)

j NH2 g 12.1835 Å

(b)

Fig. 1. (a) The chemical structure and proton assignation of SFP and (b) optimized structure with numbering system of SFP.

intramolecular charge transfer (TICT) in the first excited state as evidenced by the appearance of large Stokes shifted longer wavelength (LW) emission. The increased emission intensity of LW with a considerable bathochromic shift in CD solution has been attributed to the enhancing TICT in all the sulfanilamide molecules. Further the combination of experimental and theoretical analysis leads to successful results in solving structural, energetic and dynamic problems [14,15]. In addition, Longhi and coworkers [16,17] studied the inclusion complexes of sulfamethoxazole and sulfadiazine with b-CD and its derivatives in aqueous solution and in solid state. In this paper, we aim to report the characterization of inclusion complexes formed by SFP with a- and b-CD. We utilized absorption and fluorescence spectral techniques to determine the inclusion stoichiometry and binding constant of SFP/CD complexes. The solid complexes characterized by 1H NMR, FTIR, differential scanning calorimetry (DSC), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and powder X-ray diffraction (XRD) methods. PM3 method was also applied to study the inclusion process of the drug within CD cavity and the minimum energy structures of SFP/CD complexes in 1:1 molar ratio were proposed.

minum foil and stirred continuously for 24 h at room temperature and the solution was refrigerated overnight at 5 °C. The precipitated drug–CD inclusion complexes were recovered by filtration and washed with small amount of methanol and water to remove uncomplexed drug and CD respectively. The precipitate was then dried in vacuum at room temperature for 2 days and stored in an airtight bottle. The powder samples were analyzed by FTIR, DSC, 1 H NMR, SEM, TEM and powder XRD methods. 2.4. Preparation of physical mixture The physical mixtures of SFP and CDs were prepared in 1:1 mole ratio. The amounts were mixed by simple blending with the help of a mortar and pistol for 5 min at room temperature and then kept in an airtight bottle. The powder samples were analyzed by FTIR, DSC and powder XRD methods. 2.5. Instruments

The concentration of stock solution of the drug was 2  103 M. The stock solution (0.2 ml) was transferred into 10 ml volumetric flasks. To this, varying concentrations of a-CD or b-CD solution (1.0  103 to 1.0  102 M) was added. The mixed solution was diluted to 10 ml with triply distilled water, shaken thoroughly and kept for 6 h to bring it to a state of equilibrium. The final concentration of drug in all the flasks was 4  105 M. The experiments were carried out at room temperature (30 °C).

Absorption and fluorescence spectral measurements were carried out with Shimadzu UV–visible spectrophotometer (model 1650 PC) and Shimadzu spectrofluorimeter (model RF-5301) respectively. Fluorescence lifetime measurements were performed using a pico-second laser and a single-photon counting setup from Jobin-Yvon IBH (Madras University, Chennai, India). The fluorescence decay of the sample was analyzed using IBH data analysis software. FTIR spectra of the drug, CDs and the inclusion complexes were measured between 4000 cm1 and 400 cm1 on a JASCO FTIR-5300 spectrometer using KBr pellet with 256 scans at a resolution of 4 cm1. Thermal characteristics of solid inclusion complexes were measured using Mettler Toledo DSC1 fitted with STRe software (Mettler Toledo, Switzerland), temperature scanning range was from 25 to 210 °C with a heating rate of 10 °C/min. XRD patterns of powder samples were recorded with a BRUKER D8 Advance diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) with Cu KaI radiation (k = 1.5406 Å), a voltage of 40 kV and a 20 mA current. One-dimensional 1H NMR spectra for SFP and its CD inclusion complexes were recorded on a Bruker AVANCE 500 MHz spectrometer (Germany) using an inverse broadband (BBI) probe at room temperature. Microscopic morphological structures of the solid samples as well as SFP, a-CD and b-CD were investigated and photographed using a scanning electron microscope Hitachi S3400N. The morphological structures of drugencapsulated complexes SFP/a-CD and SFP/b-CD were investigated by TEM using TECNAI G4 microscope with an accelerating voltage of 200 kV, for the TEM analysis carbon coated copper TEM grids (200 mesh) were used.

2.3. Preparation of inclusion complexes in solid state

2.6. Molecular modeling

a-CD/b-CD (0.973/1.14 g) was dissolved in 40 ml distilled water at 40 °C in a water bath. The drug (0.249 g) in 10 ml methanol was slowly added to the CD solution with continuous agitation. The molar ratio of drug to CD was 1:1. The vessel was covered with alu-

The theoretical calculations were performed with Gaussian 03W package. The initial geometry of SFP, a-CD and b-CD were constructed with Spartan 08 and then optimized by PM3. CD was fully optimized by PM3 without any symmetry constraint. The

2. Materials and methods 2.1. Materials Sulfapyridine (SFP), a-CD and b-CD were purchased from Sigma–Aldrich chemical company and used as such. All other chemicals and solvents used were of the highest grade (Spectrograde) commercially available. Triply distilled water was used for the preparation of aqueous solutions. The aqueous solutions were prepared just before each measurement. Purity of the compounds was checked by their melting point and also by using fluorescence techniques i.e., by obtaining similar spectral profile when excited with different wavelengths. 2.2. Preparation of inclusion complexes in solutions

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3.1. Absorption spectroscopy Absorption and fluorescence spectral data of SFP in different concentrations of a-CD and b-CD are summarized in Table 1. Fig. 2 shows the absorption spectra of SFP in aqueous solutions (pH  6.5) containing varying concentrations of a-CD and b-CD. In water, three absorption bands of SFP were found at 310 nm (p ? p⁄ transition of the double bond), 261 nm (p ? p⁄ transition of the aniline group) and 243 nm (p ? p⁄ transition of the aromatic ring). With increasing concentration of b-CD, the absorption maxima of SFP are red shifted (about 7 nm) with gradual decrease in the molar extinction coefficient whereas in a-CD the absorbance increased at the same wavelength. These results indicate that SFP is entrapped into the CDs cavities to form stable inclusion complexes. Further, the red shift observed in b-CD reveals that nitrogen atom of pyridine ring interacts with the secondary hydroxyl groups of b-CD, because it is well known that CDs are good hydrogen donors. Additionally, clear isosbestic points (at 280 and 325 nm) were observed in the absorption spectra. In general, the existence of an isosbestic point in the absorption spectra is an indication of the formation of well defined 1:1 inclusion complex [18,19].

0.41

Absorbance

7

0.37 Abs 0.33 0.29 1 0 0.40

0 200

310 nm 4 8 12 [α-CD] × 10-3 M

300

400

Wavelength (nm) 0.80

(b)

0.39

310 nm

0.35

Abs 0.31 0.27

1

0

0.40

4 8 12 [β-CD] × 10-3 M

7 7 1

0 200

300

400

Wavelength (nm) Fig. 2. Absorption spectra of SFP in different (a) a-CD and (b) b-CD concentrations (M): (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.01. Insert Fig.: absorbance vs. CD concentration.

300

Fluorescence intensity (au)

3. Results and discussion

(a)

0.80

Absorbance

glycosidic oxygen atoms of CD were placed onto the XY plane and their center was defined as the center of the coordination system. The primary hydroxyl groups were placed pointing toward the positive Z axis. The inclusion complexes were constructed from the PM3 optimized CD and guest molecules. The longer dimension of the guest molecule was initially placed onto the Z axis. The position of the guest was determined by the Z coordinate of one selected atom of the guest. Complete geometry optimizations without any restriction with PM3 method were employed in the study of the complexation process of the drug within a-CD and b-CD. The most energetically favorable structures of the drug as well as CD molecules were used to construct CD inclusion complexes. These drug–CD complexes were constructed by manually inserting the drug into the CD cavity through the wider rim, centering it on a vector perpendicular to the mean plane through the glycosidic oxygen atoms.

(a)

120

310 nm 50 0

7 150

8 12 4 [α-CD] × 10-3 M

1

0 320

3.2. Fluorescence study

190

If

450

580

Wavelength (nm)

Table 1 Absorption and fluorescence maxima (nm) of SFP with different concentrations of aCD and b-CD. Concentration of CD (M)

0 (without CD)

0.006

0.010

Excitation wavelength (nm) K (M1) DG (kcal mol1)

a-CD

b-CD

kabs

log e

kflu

kabs

log e

kflu

310 261 243 310 261 243 310 261 243 310

3.52 4.21 4.18 3.54 4.23 4.20 3.55 4.24 4.21

348 371s 434 348 371s 434 348 434

310 261 243 311 265 244 314 268 243 310

3.52 4.21 4.18 3.48 4.17 4.14 3.46 4.15 4.12

348 372s 434 348s 380 440 386 445

227 3.26

355 3.53

301 3.43

789 4.01

300

Fluorescence intensity (a.u)

Fig. 3 displays the fluorescence spectra of SFP in aqueous solution as a function of a-CD and b-CD concentrations. The effect of CDs on the emission spectra of the SFP molecules are more pronounced than the corresponding effect on the absorption spectra

(b)

300 If 200 100 0

7 150

0 320

434 nm 0

1

450

4 8 12 [β-CD] × 10-3 M

580

Wavelength (nm) Fig. 3. Fluorescence spectra of SFP in different (a) a-CD and (b) b-CD concentrations (M): (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.01. Insert Fig.: fluorescence intensity vs. CD concentration.

with respect to the concentration of CDs. SFP emits two emission maxima at 348 and 434 nm with a shoulder at 372 nm when excited at 310 nm. It has been explain in our earlier studies [11,12] that sulfonamide derivatives undergo normal as well as highly

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Stokes shifted fluorescence. The normal fluorescence takes place from the locally excited (LE) state while the twisted intramolecular charge transfer (TICT) is responsible for highly Stokes shifted fluorescence. The PM3 calculations have suggested that the large stabilization of excited singlet state of SFP with twisted conformation occurring at the amide SAN bond between the electron donor group (aniline ring) and the electron acceptor group (pyridine ring). However, Rajendiran et al. [20] reported whenever two aromatic rings are separated by the groups like ASO2, ACH2, ACO and ANH, they can form a TICT state. Thus, it can be speculated that the enhanced 434 nm in the SFP drug emission should originate from the TICT state. In aqueous solution, the LE band intensity is greater than TICT band. With an increasing the concentration of b-CD, a regular red shift was observed in the TICT band (434–445 nm) and the shoulder at 372 nm appeared the same trend but the SW band appeared blue. In contrast to b-CD, the position and shape of the LE and TICT bands were not changed in aCD. However, on addition of a-CD, both the LE and TICT intensities were equally increased. The enhancement of the fluorescence intensities of both LE and TICT emission bands may be due to lowering of solvent polarity provided by CD cavity and the non-radiative path of LE emission is restricted through TICT state [11,12]. Further, the enhancement of TICT emission is observed in both CDs suggesting that the inclusion process plays the major role in the TICT emission. This inference is supported by similar emission spectra observed when excited CD solutions containing sulfa drug with different emission wavelengths at 340 and 440 nm respectively [11,12]. The presence of isosbestic point in the absorption spectra suggested that SFP formed 1:1 inclusion complex with a-CD and bCD. The K value for the formation of 1:1 inclusion complexes can be evaluated from Benesi–Hildebrand (B–H) double reciprocal plots concerning the absorption and fluorescence intensity changes [21]. Fig. S1 illustrates the double reciprocal plots for the drug containing the CDs. A good linear relationship is obtained when 1/ (I  I0) is plotted against 1/[CDs], indicating that the stoichiometry of the complexes is 1:1. The binding constant (K) for the inclusion complex formation as obtained from the slope and intercept of the double reciprocal plots was found to be SFP/a-CD: abs  227 M1, flu  355 M1 and SFP/b-CD: abs  301 M1, flu  789 M1. The variation in the association constants suggested that b-CD has greater inclusion ability than a-CD. The negative free energy change (DG) values of the complexes suggest that the binding process is a spontaneous (Table 1). 3.3. Time-resolved fluorescence analysis In order to substantiate the interpretation of steady-state spectroscopic measurement data, the time-resolved fluorescence spectra of the drug (at 340 nm) as a function of real time in the absence or presence of 0.01 M a-CD and b-CD were analyzed. The relevant data are compiled in Table 2 and the decay curves are shown in Fig. S2. These data indicate that the significant improvement of the stability of the drug upon addition of CDs. Further, the results proved that b-CD gives better stabilization compared with a-CD. The fluorescence decay curve for the free drug (SFP) in water was fitted to biexponential function with v2 values of 1.1. This reveals Table 2 Fluorescence decay parameters of SFP in water and 0.01 M CD solution. Medium

Water a-CD b-CD

Lifetime (ns)

Pre-exponential factor

s1

s2

s3

a1

a2

a3

0.27 0.36 0.19

3.67 1.82 2.72

5.21 8.75

0.29 0.27 0.22

0.04 0.08 0.07

0.03 0.01

hsi

2.48 2.79 4.03

that the drug has two lifetime components. The first one with a larger lifetime is assigned to LE emission and the other one is TICT state of SFP molecule. It is noteworthy that the decay time of the slow component is different to that of TICT emission within experimental uncertainty. This suggest that a little equilibrium between the locally excited (LE) state and the TICT state is achieved in water in a rather short period. It has to be pointed out here that the fluorescence decay of SFP in water analyzed individually is not well reproduced by a double exponential. By the addition of CDs, biexponential decay curve becomes triexponential. Three lifetimes (s1, s2 and s3) were obtained in the presence of both a-CD and b-CD. The data in Table 2 reflect the observed lifetime values increase with the addition of CD concentration in aqueous solution, due to the inclusion complex formation between the drug and CDs. The observed enhancement in lifetime indicating that SFP molecule experiences less polar hydrophobic environments within the CD cavity resulting non-radiative decay processes were reduced [14,15]. Thus, the increase in fluorescence lifetimes is a result of the significant interactions of the sulfa drug with hydrophobic CD nanocavity. The efficient lifetime enhancement is found to be higher with b-CD suggests that the presence of stronger hydrophobic interaction between the SFP and b-CD. 3.4. Possible inclusion complex formation From the above discussions, the possible inclusion mechanism is proposed as follows: According to the molecular dimension of SFP (long axis 12.1835 Å, Fig. 1b), it is too large to fit entirely in the CD cavity and the entire guest molecule cannot fully entrapped in the hydrophobic cavity. Since, the internal diameter of a-CD and b-CD was found to be approximately 5.6 Å and 6.5 Å respectively and both CDs height is 7.8 Å. Therefore, naturally two different types of inclusion complex formation between SFP and CDs are possible: (i) aniline ring is captured in the CDs cavities and (ii) pyridine ring is captured in the CDs cavities. In b-CD, the monocation maximum of SFP (protonation of tertiary nitrogen atom) is red shifted than aqueous medium. This results indicating that the heterocyclic ring interacts with the CD hydroxyl groups [21,22]; i.e., pyridine ring is encapsulated in the CD cavity. Further, in this complex, the CD cavity will impose a restriction on the free rotation of the pyridine group in the excited state. In this type of inclusion, TICT emission should increase in CDs medium. Further, SFP does not exhibit TICT emission in non-polar solvents due to a weak dipole–dipole interaction between RASO2ANHA group with solvent and relatively fast back charge transfer. These features support the idea that the TICT state in CDs is stabilized through complexation. This confirms that the environments around the aniline group in CD medium are same as in the bulk aqueous medium and the pyridine ring is present inside of the CD cavity. 3.5.1. FTIR spectral analysis Inclusion complexation of SFP into CD cavity was confirmed by FTIR spectroscopy [23,24] because the bands resulting from the inserted part of the guest molecule are generally shifted or their intensities changed. FTIR spectra of SFP, physical mixtures (1:1 mole ratio) and the solid inclusion complexes are shown in (Supplementary Fig. S3). The amino, amido and CH stretching frequencies of SFP at 3418 cm1, 3310 cm1 and 3244 cm1 respectively were merged and moved in the inclusion complexes to 3379 cm1 and 3383 cm1. The C@C bending frequencies of SFP at 1637 cm1 and 1583 cm1 is moved in the inclusion complexer to 1643 cm1 and 1585 cm1. The SO2 stretching frequency appears at 1367 cm1 was shifted in the inclusion complexes to 1370 cm1 and the intensities were also decreased. Further, the frequency ranges of SFP from 947 cm1 to 1244 cm1 was also shifted in the inclusion complexes. However, the CANH2 stretching

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219

appeared at same frequency in the inclusion complexes, confirming that the amino group is not included in the CD cavity. The above changes could be due to the formation of SFP/CD inclusion complexes in solid state. 3.5.2. Differential scanning calorimetry (DSC) The thermal properties of SFP/a-CD and SFP/b-CD complexes were analyzed by DSC, which represents an effective and inexpensive analytical tool for an accurate physicochemical characterization of drug–CD inclusion complexes in the solid state. This analysis is widely used for a rapid preliminary qualitative investigation of the thermal behavior of the single components, their physical mixtures and the inclusion complexes [16,25]. The DSC thermograms of SFP, a-CD, b-CD, physical mixtures and the solid inclusion complexes are presented in Fig. 4. The thermal curve of SFP was typical of a crystalline anhydrous substance with a sharp endotherm at 191.6 °C indicating the melting point of the drug. The DSC profile of a-CD showed three endothermic peaks around at 79, 109, 137 °C whereas a broad endothermic peak at 128 °C was observed for b-CD. These endothermic effects are mostly associated to water losses from CD cavities. In the physical mixtures, the intensity of the peak corresponding to the melting point of the drug is reduced and its position slightly displaced. On contrary, the complete disappearance of characteristic melting peak of SFP in the DSC curves of the inclusion complexes is an indicative of a strong interaction between the drug and CD. However, the endotherms of a-CD and b-CD were shifted to 67.4, 107.1, 124.5 °C and 116.8 °C respectively, accompanied by significant decrease in the intensity. These results further confirmed the formation of SFP/CD inclusion complexes with different properties. The shifts in thermal features of CD to lower temperature in the complex may be due to the reorientation of CD by the insertion of the drug molecules. 3.5.3. X-ray diffraction (XRD) analysis XRD patterns of SFP, a-CD, b-CD and their inclusion complexes are illustrated in Fig. 5. The XRD pattern of a-CD showed the characteristic peaks at 2h values of 9.46°, 11.82°, 14.19°, 17.93°, 21.57° and 27.12°. However, the diffraction pattern of b-CD crystals exhibited the important peaks at 2h values of 8.95°, 10.56°, 12.46°, 18.75°, 22.57°, 27.03°, 31.86° and 34.59°. SFP showed the characteristic peaks at 2h value of 11.10°, 13.37°, 14.65°, 15.38°, 15.74°, 19.29°, 21.75°, 22.66°, 24.57°, 25.76°, 30.04°, 34.59° and 35.59°, indicating the higher degree of crystallinity. Most of the characteristic peaks of SFP were present in the diffraction patterns of physical mixtures of the drug with CDs without loss of intensity. In contrast, the XRD of the SFP/a-CD and SFP/b-CD complexes

2

Exothermic (mW/mg)

0 -2 -4 -6

Pure SFP Pure α-CD Pure β-CD SFP/α-CD SFP/β-CD

-8 -10 40

60

SFP/α-CD mixture SFP/β-CD mixture

80

100

120

14 0

160

180

200

Temperature (oC) Fig. 4. DSC curves of SFP, pure a-CD, pure b-CD, SFP/a-CD mixture, SFP/b-CD mixture, SFP/a-CD complex and SFP/b-CD complex (25–210 °C at 10 °C/min).

Fig. 5. Powder XRD patterns of (a) SFP, (b) a-CD, (c) b-CD, (d) SFP/a-CD complex and (e) SFP/b-CD complex.

(Fig. 5d and e) showed amorphous halo patterns, which were quite different from the superimposition of crystalline a-CD or b-CD and the drug, indicating the formation of the inclusion complex between a-CD or b-CD and SFP [17,26]. Additionally, most of the crystalline diffraction peaks of CDs disappeared after complexation with SFP, indicating that the complexation of SFP reoriented in the CD molecules to some extent. Further, the less sharp peaks in the XRD of the SFP/CD complexes indicate that the solid complexes possess a more amorphous structure than the isolated drug and CDs. 3.5.4. 1H NMR analysis Further evidence for the formation of inclusion complexes can be obtained from the changes of chemical shifts of SFP or CDs in 1 H NMR spectroscopy, which has proved to be an useful analytical tool in the study of CD inclusion complexes [15,27]. The 1H NMR spectra and proton numbering of SFP is presented in Fig. S4. The chemical shifts values for the isolated drug and the inclusion complexes are given below: SFP/a-CD complex/ b-CD complex: Ha  11.108/11.072/11.065; Hb  8.092/8.097/8.096; Hc  7.717/ 7.723/7.722; Hd  7.519/7.503/7.492; He  7.069/7.052/7.043; Hf  6.898/6.902/ 6.904; Hg  6.547/6.531/6.526; Hj  5.961/ 5.948/5.946. The majority of the drug protons showed a down-field shift in the presence of CDs, while only Hb and Hc protons were shifted to up-field. The d value of Ha is the amino group (ANH2) protons of SFP shifted to 0.036 ppm in the a-CD complex and 0.043 in the b-CD complex. Significantly, the aromatic protons of the SFP molecule (Hd, He and Hg) also shifted up field in the inclusion as compared with the free drug (Supplementary Fig S4). These results indicate that the aromatic protons strongly interact with CD inner protons. The down-field shift may be related to changes in the local polarity due to the inclusion of SFP drug into the hydrophobic cavity of CDs or to the deshielding effects caused by the van der Waals

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interaction between the SFP and CD cavity. Based on these results, it may be assumed that the part of aniline moiety and pyridine ring were included into the center of CD cavity. To further explore the inclusion mode, the chemical shifts of CD protons in the absence and presence of SFP were investigated. The resonance assignment of CD protons are well established and consists of six types of protons, the chemical shift of CD protons reported in this work is in agreement with those reported by different authors [27]. The insertion of SFP into the CD cavity caused an upfield shift of CD protons. As can be seen from 1H NMR spectra, the inclusion complexation with the drug had a negligible effect on the d values of H-1, H-2, H-4 and H-6 protons of CD (0.007 ppm). In contrast, those values of H-3 and H-5 protons which are located in the interior of the cavity exhibited significant changes (0.032–0.048 ppm). It is fairly noteworthy that H-3 protons shifted (0.038 ppm), but H-5 protons showed weak shift (0.031 ppm) after the formation of inclusion complex. Because both H-3 and H-5 protons are located in the interior of CD cavity, and H-3 protons are nearer to the wider side of cavity while H-5 protons are nearer to the narrow side, this phenomenon may indicate that SFP should penetrate deeply into the CD cavity from the wider rim side. 3.5.5. Scanning electron microscopy (SEM) Imaging of inclusion complexes by SEM is expected to provide information on the surface morphology and size. In this regard, first we captured the microphotographs of the powder form of the drug, a-CD and b-CD by SEM and then we also captured powder form of the inclusion complexes (Fig. 6). These photographs clearly elucidated the difference of crystal state between the pure components and their inclusion complexes. Examination of SEM photographs of SFP showed micronized crystals, compact structures can be observed with irregular shapes and different sizes, characterized by smooth surface and obtuse corners. a-CD presented prismatic crystals with well-developed face whereas plate shaped crystals were observed for b-CD. However, the morphology of the inclusion complexes is quite different in shape and size from pure SFP and CDs. In the solid complexes, the original morphology of the raw materials disappeared and it was not possible to distinguish the solid samples from the starting materials. Obviously, the SFP/a-CD complex exhibited bar like shaped crystals whereas

irregular shaped crystals were observed for SFP/b-CD complex. Once again, the data obtained from SEM are supported to previous results, suggesting the inclusion complex formation between the SFP and CDs. In addition both solid crystals showing cracks and dimples on the surface, confirming that the coprecipitation method is able to encapsulate the SFP into the CD cavity. 3.5.6. Transmission electron microscopy (TEM) analysis TEM is utilized for visualizing the surface morphology and size of the aggregates of nano-encapsulated sulfa drug in water at room temperatures [28,29]. TEM analysis is an ultimate for quantitatively visualizing the surface texture of the substance. TEM images of SFP/a-CD and SFP/b-CD complexes are shown in Fig. 7. For the both inclusion complexes, round (presumably spherical) shaped nanoparticles with the average size about 20–50 nm were observed. A similar guest encapsulation associated with nanoparticle formation was found for another hydrophobic drug molecule [30]. Preparation of nanoparticles from preformed inclusion complexes of the sulfa drugs and a-CDor b-CD proved to be an effective method to enhance drug loading to nanoparticles. These results suggested that the CD may serve as delivery carrier for hydrophobic drugs. 3.6. Molecular modeling studies In order to rationalize the experimental results described above, we carried out molecular modeling studies of the complexes using semiempirical quantum mechanical calculations (PM3 method) [31,32]. The optimized geometry of the inclusion complexes with the lowest bind energy is depicted in Fig. S5. Molecular modeling by PM3 method established that the geometry of the inclusion complexes in well agreement with the experimental data. As a result of this calculation, it was established that the part of SFP molecule is included into the CD cavity, with pyridine ring oriented toward the secondary rim, whereas the aromatic ring is oriented to the primary rim of the cavity. But the NH2 group is projected outside of the hydrophobic CD cavity. The optimization at PM3 level of theory revealed that the geometry of SFP molecule is not a plane; i.e., the structure bends at SO2ANH group (Fig. 1b). If they form a 1:2 inclusion complex, the two CD molecules are very close to each other, causes the repulsion between the two CD molecules

Fig. 6. Scanning electron microphotographs of (a) a-CD, (b) b-CD, (c) SFP, (d) SFP/a-CD complex and (e) SFP/b-CD complex.

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Fig. 7. TEM images of (a) SFP/a-CD and (b) SFP/b-CD nanoparticles.

and the CD molecules becoming farther and farther from each other. This is observed in the simulation process by PM3 method. Table S1 summarizes the calculated energy, free energy, enthalphy, entropy, dipole moment, HOMO and LUMO for the optimized geometries of SFP/CD (1:1) inclusion complexes along with the isolated drug and CD molecules. The PM3 calculations express that the energies of the complexation are lower than that of the isolated host and guest molecules. The above values allowed us to evaluate the inclusion process and to find the most stable inclusion complex between the complexes under study. It is clear from Table S1 that the complexation process is energetically favorable in nature. The large negative formation energy upon complexation clearly demonstrated that the b-CD could form stable complexes with SFP than that of a-CD. We also computed the dipole moment of the 1:1 inclusion systems. Significantly, the inclusion complexes (l 6 10.30 D) showed higher dipole moment values than the free drug molecule (4.66 D). This indicates that the polarity of the CD changed after the drugs entered in to the CD cavity. From these results it can be concluded that the dipole moment value shows a strong correlation with the complexation behavior of the guest molecules. Further, the optimized inclusion structures in Fig. S5 demonstrated the hydrogen bonds are formed in the inclusion complexes (shown as black dotted line). Based on the results, it was found that there are three hydrogen bonds between oxygen atoms of SO2 group and nitrogen atom in the pyridine moiety of SFP and hydrogen atoms (both H-3 and H-5) of the CD cavity with a dH  O or dH  N distance less than 3.0 Å [33]. This justified the importance of both interaction energy between the drugs and CD necessary to ensure a better inclusion of the guest to the host. The above values were supported by the fact that the flexibility of the host molecule may be one of the structural requirements for the inclusion complex formations. The present calculations explained that hydrogen bonding brings the difference between the binding energies of both a-CD and b-CD complexation with the sulfa drug. However a careful comparative analysis of the energetic values suggested that the mutual host-guest hydrogen bonding interactions contribute greatly to complex formation energy and are crucial in determining stability of the complexes. The overall PM3 calculation results are in good agreement with that of the experimental results. 4. Conclusions The inclusion complexation behavior of SFP with a-CD and b-CD were investigated using absorption, fluorescence, time-resolved fluorescence, FTIR, DSC, XRD, 1H NMR, SEM, TEM and molecular modeling methods. The enhancement of TICT emission in both CDs suggesting that the inclusion process plays the major role in the TICT emission. The spectral shifts revealed that part of pyridine

ring of SFP is entrapped in the inside of CDs cavities. TEM images confirmed round shaped nanoparticles with the average size about 20–50 nm were observed in SFP with a-CD and b-CD inclusion complexes. PM3 calculations have suggested that the large stabilization of excited singlet state of SFP with twisted conformation occurring at the amide SAN bond between the electron donor group (aniline ring) and the electron acceptor group (pyridine ring). Acknowledgements This work is supported by the CSIR [No. 01(2549)/12/EMR-II] and UGC [F. No. 41-351/2012 (SR)] New Delhi, India. One of the authors S. Siva is thankful to UGC, New Delhi for the award of Senior Research Fellow (SRF) through RGNF Scheme (No. F. 161281(SC)/2009(SA-III)). We are grateful to Dr. R. Chandrasekar, School of Chemistry, University of Hyderabad for his kind help and UGC Networking Resource Centre, School of Chemistry, University of Hyderabad for providing instrumental facilities. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molstruc.2013.09. 035. References [1] A. Biwer, G. Antranikian, E. Heinzle, Appl. Microbiol. Biotechnol. 59 (2002) 609–617. [2] F. Vogtle, Supramolecular Chemistry: An Introduction, John Wiley and Sons Ltd., New York, 1991. [3] J. Szejtli, Chem. Rev. 98 (1998) 1743–1753. [4] S. Li, W.C. Purdy, Chem. Rev. 92 (1992) 1457–1470. [5] T. Loftsson, M.E. Brewster, J. Pharm. Sci. 85 (1996) 1017–1025. [6] V.J. Stella, R.A. Rajewski, Pharm. Res. 14 (1997) 556–567. [7] T. Loftsson, T. Jarvinen, Adv. Drug Deliv. Rev. 36 (1999) 59–79. [8] J. Marek, FarmakoterapieVnitrnichNemoci (Pharmacotherapy of Internal Diseases), Grada Publishing, Prague, 1998. p. 159. [9] A.M. Jacobsen, B. Halling-Sørensen, F. Ingerslev, S.H. Hansen, J. Chromatogr. A 1038 (2004) 157–170. [10] Z.X. Cai, Y. Zhang, H.F. Pan, X.W. Tie, Y.P. Ren, J. Chromatogr. A 1200 (2008) 144–155. [11] J. Premakumari, G. Allan Gnana Roy, A.A.M. Prabhu, G. Venkatesh, V.K. Subramanian, N. Rajendiran, Phys. Chem. Liq. 49 (2011) 108–132. [12] A.A.M. Prabhu, G. Venkatesh, N. Rajendiran, J. Solution Chem. 39 (2010) 1061– 1086. [13] G. Venkatesh, T. Sivasankar, M. Karthick, N. Rajendiran, J. Incl. Phenom. Macrocycl. Chem. (2013), http://dx.doi.org/10.1007/s10847-012-0248-z. [14] T. Sivasankar, A.A.M. Prabhu, M. Karthick, N. Rajendiran, J. Mol. Struct. 1028 (2012) 57–67. [15] A.A.M. Prabhu, R.K. Sankaranarayanan, G. Venkatesh, N. Rajendiran, J. Phys. Chem. B 116 (2012) 9061–9074. [16] A. Delrivo, A. Zoppi, M.R. Longhi, Carbohydr. Polym. 87 (2012) 1980–1988. [17] C. Garnero, V. Aiassa, M.R. Longhi, J. Pharm. Biomed. Anal. 63 (2012) 74–79. [18] T.H. Kim, D.W. Cho, M. Yoon, J. Phys. Chem. 100 (1996) 15670–15676.

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