WITHDRAWN: Investigation of α- and β-cyclodextrin inclusion complexes with mefenamic acid and aceclofenac drugs: Spectral and theoretical study

MOLLIQ-04041; No of Pages 13 Journal of Molecular Liquids xxx (2014) xxx–xxx

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Investigation of α- and β-cyclodextrin inclusion complexes with mefenamic acid and aceclofenac drugs: Spectral and theoretical study N. Rajendiran ⁎, T. Mohandass, J. Thulasidhasan Department of Chemistry, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 10 August 2013 Received in revised form 23 November 2013 Accepted 25 November 2013 Available online xxxx Keywords: Mefenamic acid Aceclofenac drug Cyclodextrin Inclusion complex Nanoparticles TEM Molecular modeling

a b s t r a c t Inclusion complexes of mefenamic acid (MFA) and aceclofenac (ALF) with α- and β-cyclodextrins in aqueous medium were investigated by absorption, fluorescence and time resolved fluorescence methods. The solid inclusion complexes between CDs and drugs were characterized by SEM, TEM, FT-IR, 1H NMR, DSC and XRD techniques. Spectral studies indicated that both CDs form 1:1 inclusion complex with MFA and ALF. The experimental results revealed that the inclusion process is a spontaneous process. Time-resolved fluorescence studies suggested that ALF exhibited biexponential decay in aqueous and triexponential decay in CD whereas significant enhancement of lifetime of decay components of MFA was observed. Morphologies of drug–CD complexes observed by TEM demonstrate that self-aggregates of MFA/α-CD, ALF/α-CD and ALF/β-CD were nano-sized particles while vesicles were observed for MFA/β-CD. A spatial arrangement of inclusion complex is proposed based on 1H NMR and PM3 results. Investigations of thermodynamic and electronic properties confirmed the stability of the inclusion complex. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Inclusion complexation and molecular recognition are of current interest in host–guest chemistry or supramolecular chemistry [1,2]. Cyclodextrins (CDs) are cyclic oligosaccharides of α-(1,4) linked D-glucose units in a ring formation containing a relative hydrophobic central cavity and a hydrophilic outer surface. The most common CDs are α-, β-, and γ-CDs and are formed by six, seven, and eight glucose units, respectively. These CDs generate a hollow truncated cone structure because of the 4C1 chair conformation of the sugar units [3]. The height of the CD cavities is 7.9 Å, where the bottom and top diameters are 4.7 and 5.3 Å for α-CD, 6.0 and 6.5 Å for β-CD, and 7.5 and 8.3 Å for γ-CD, respectively [2]. One of the key chemical properties of cyclodextrins is their ability to form inclusion complexes with a wide variety of molecules that accommodates a guest molecule into their inner cavity. Inclusion complexes have been extensively used to improve the solubility, stability and bioavailability of various drugs [4]. They are molecular compounds, having the characteristic structure of adducts, in which one of the host molecules spatially encloses another one. The guest molecule is located in the cavity of the host molecule without affecting the framework structure of the host [2,5]. These include alteration of the solubility of the guest compound, stabilization against the effects of light, heat, and oxidation, masking of unwanted physiological effects,

⁎ Corresponding author. Tel.: +91 94866 28800 (Mobile); fax: +91 4144 238080. E-mail address: [email protected] (N. Rajendiran).

reduction of volatility, and others. In some applications, more benefits are obtained by complexation with cyclodextrins [6]. Non-steroidal anti-inflammatory (NSAI) drugs are a group of drugs of diverse chemical composition and different therapeutic potentials having a minimum of three common features: identical basic pharmacological properties, similar basic mechanism of action as well as similar adverse effects. Most NSAI drugs are metabolized in the liver by oxidation and conjugation to inactive metabolites which are typically excreted in the urine, although some drugs are partially excreted in bile. Metabolism may be abnormal in certain disease states, and accumulation may occur even with normal dosage [7]. Mefenamic acid (2-[(2,3dimethylphenyl)amino]benzoic acid, MFA) (Fig. 1) belongs to a family of NSAI drugs with antipyretic and strong analgesic properties which is a derivative of N-phenylanthranilic acid. MFA is used for the treatment of joint disorders and various kinds of pain such as headache, dental pain, and post-operative and post-partum pain. It has been widely applied in the pharmaceutical field [8]. Aceclofenac (2-[(2, 6dichlorophenyl) amine] phenylacetoxyacetic acid, ALF) is an orally effective NSAI drug of the phenyl acetic acid group. It possesses antiinflammatory, analgesic and anti-pyretic activities [8]. ALF drug is used for the treatment of rheumatoid arthritis and is selected as a model drug. However both drugs exhibit low bioavailability related to its poor water solubility. The bioavailability enhancement of NSAI drugs in water is an important goal and it is hoped that their complexation with suitable hosts may allow for the development of analytical techniques and remediation procedures. Recently various studies involving inclusion complexation

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N. Rajendiran et al. / Journal of Molecular Liquids xxx (2014) xxx–xxx

a 2.510 Å

B

A

Conformer I (closed)

b

B

A

Conformer II (open)

2.636 Å

B

A

Conformer I (closed)

B

A

Conformer II (open)

Fig. 1. The B3LYP/6-31 + G optimized ground state geometrical structures of (a) MFA and (b) ALF with (closed conformer I) and without (open conformer II) intermolecular hydrogen bonding (red color—oxygen atom; blue—nitrogen atom; green—chlorine atom). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

phenomena have been reported for new generation NSAIDs which are almost insoluble in water, primarily to enhance the oral bioavailability and dissolution behavior [9,10]. The aims of this work are to establish the complexation behavior of these NSAI drugs with cyclodextrins in aqueous media, and to determine the structure of these inclusion complexes. Changes in UV-absorption and fluorescence spectra were quantitatively measured for MFA and ALF with α-CD and β-CD to obtain the corresponding binding constants. The solid complexes were characterized by SEM, TEM, FT-IR, 1H NMR, DSC and XRD techniques. The schematic representation of this inclusion process is proposed by molecular modeling studies (using PM3 method). 2. Experimental 2.1. Reagents and materials MFA, ALF, spectrograde solvents, α-CD and β-CD were purchased from Sigma-Aldrich chemical company and used as such. Purity of the compound was checked by melting point and also by using fluorescence techniques i.e., by getting same spectral profile when excited with different wavelengths. Triply distilled water was used for the preparation of aqueous solutions. 2.2. Preparation of inclusion complexes in solution A stock solution of drug was prepared in methanol and the concentration of the stock solution was 2 × 10−3 M. Exactly 0.2 ml of this stock solution was transferred into each of the 10 ml volumetric flasks. To this, varying concentrations of α-CD or β-CD solution (range from 1.0 × 10− 3 to 1.2 × 10− 2 M) were added. The mixed solution was diluted to 10 ml with triply distilled water and shaken thoroughly.

The final concentration of the drug in all the flasks was 4 × 10−5 M. The aqueous solutions were prepared just before each measurement. 2.3. Preparation of solid inclusion complexes α-CD/β-CD (0.973/1.14 g) was dissolved in 40 ml distilled water at 40 °C in a water bath. The drug (0.241 g for MFA or 0.354 g for ALF) 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 aluminium foil and stirred continuously for 24 h at room temperature and the solution was refrigerated overnight at 5 °C. The precipitated drug–α-CD and drug–β-CD 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 two days and stored in an airtight bottle. 2.4. Instruments Absorption and fluorescence spectral measurements were carried out with a Shimadzu UV–visible spectrophotometer (model 1601 PC) and Shimadzu spectrofluorimeter (model RF-5301), respectively. The fluorescence lifetime measurements were performed using a pico-second laser and a single-photon counting setup from Jobin-Yvon IBH (Madras University, Chennai, India). A diode-pumped Millena CW laser (Spectra Analysis) at 532 nm was used to pump the Ti-sapphire rod in a Tsunami picosecond mode-locked laser system (Spectra Physics, model 4690 M3S). The Ti-sapphire rod was oriented at Brewster's angle to the laser beam. The wavelength turning range was 280–540 nm (i.e., standard pico configuration). The fluorescence decay of the sample was analyzed using IBH data analysis software. The SEM photographs were collected on a Hitachi S3400N. The morphologies of nano-encapsulated complexes

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of MFA/CD and ALF/CD were investigated by TEM using a TECNAI G4 microscope with an accelerating voltage of 200 kV, and for the TEM analysis carbon coated copper TEM grids (200 mesh) were used. FT-IR spectra of powder sample of drugs, α-CD, β-CD and the inclusion complexes were measured between 4000 cm−1 and 400 cm−1 on a JASCO FT-IR-5300 spectrometer using KBr pellet with 256 scans at a resolution of 4 cm−1. One-dimensional 1H NMR spectra for MFA, ALF and its CD inclusion complexes were recorded on a Bruker AVANCE 500 MHz spectrometer (Germany) using an inverse broadband (BBI) probe at room temperature. Samples were dissolved in DMSO-d6 (99.98%) and were equilibrated for at least 1 h. 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 180 °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 KαI radiation (λ = 1.5406 Å), a voltage of 40 kV and a 20 mA current. 2.5. Computational method Molecular modeling of the inclusion complexes was carried out using Gaussian 03 W program. Considering the large number of atoms in the studied systems, an adequate choice of the modern chemistry had to inevitably result from a compromise between the available computer power and the desired level of calculation. The compromise was particularly important for full geometry optimization calculations were computationally expansive at the ab initio level for such large system. Because the semiempirical PM3 method has been shown to be a powerful tool in the conformational study of CD complexes [11,12] and has high computational efficiency in calculating CD system, it was selected to study the inclusion process. The initial geometries of MFA, ALF, α-CD and β-CD were constructed, with Spartan 08 and then optimized by the PM3 method. The glycosidic oxygen atoms of CDs were placed onto the XY plane and their center was defined as the center of the coordination system. The guest molecule was initially placed on to

a) pH~1

3. Results and discussion 3.1. Effect of α- and β-cyclodextrins Figs. 2 to 4 display the absorption and fluorescence spectra of MFA and ALF (4 × 10−5 M) in pH ~ 1 and pH ~ 7 solutions containing different concentrations of α-CD and β-CD and the spectral maxima are given in Tables 1 and 2. At pH ~ 7, both NSAI drugs exist as a carboxylic anion, hence we also recorded the spectrum for neutral form at pH ~ 1. In the absence and presence of CD concentrations, the absorption spectral shape of ALF is similar in both buffer solutions except in β-CD at pH ~ 7 (Fig. S1, Supplementary data). However in MFA, it was different at both pH solutions. The absorption maximum of MFA appears at 354 nm in pH ~ 1 whereas, in pH ~ 7 the absorption maximum appears at around 335 nm. Upon increasing the β-CD concentrations the absorption maxima of MFA in pH ~ 7 solution are seen to undergo a marginal red shift (354 nm), while a regular red shift was observed with increasing concentrations of α-CD. Nevertheless no significant spectral shift was observed in pH ~ 1 solutions consisting both CDs. However, in both solutions the absorbance enhanced with increasing CD concentrations. In the case of ALF, a new peak at 220 nm in β-CD solutions at pH ~ 7 (Fig. S1d) was observed. The above experimental results revealed that both drug molecules are transferred from more protic environments to less polar CD nanocavity [13–15]. Interestingly at higher CD concentrations (10 × 10−3 M), the absorption maxima and spectral shape of the drug molecules in both pH ~ 1 and pH ~ 7 solutions are the same, which suggests that a similar type of inclusion complexes is formed. In both cases, the presence of clear isosbestic points in the absorption spectra indicates that well defined 1:1 inclusion complex is formed between the drug and CD molecules.

1.20

0.25 Abs 0.22 0.19

0.50

the Z-axis. The positions of the MFA and ALF molecules were determined by Z-coordinate. We optimized two possible orientations: (i) aromatic ring-A inserted into the center of CD cavity, and (ii) aromatic ring-B inserted into the center of CD cavity.

364 nm 0 5 10 15 [α-CD] × 10-3 M

7

Absorbance

Absorbance

1.00

3

b) pH~7

335 nm 0.22

0.60

1

300

0 200

400

Wavelength (nm) 1.60

0.32 Abs 0.25

c) pH~1

0.18

0.60

d) pH~7 364 nm 0 5 10 15 [β-CD] × 10-3 M

7

300

Wavelength (nm)

0.40 Abs 0.31 335 nm 0.22

0.80

0 5 10 15 [β-CD] × 10-3 M

7

1 0 200

400

300

Wavelength (nm)

Absorbance

Absorbance

1.20

0 5 10 15 [α-CD] × 10-3 M

7

1 0 200

0.32 Abs 0.27

1

400

0 200

300

400

Wavelength (nm)

Fig. 2. Absorption spectra of MFA in different α-CD and β-CD concentrations (M) at pH ~1 and pH ~7: (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.010. Inset Fig.: absorbance vs. CD concentration.

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600

520 If 450

a) pH ~1

442 nm 380

300

0

7

10 15 5 [α-CD] × 10-3 M

1

0 375

487.5

Fluorescence intensity (a.u)

N. Rajendiran et al. / Journal of Molecular Liquids xxx (2014) xxx–xxx

Fluorescence intensity (a.u)

4

600

400

436 nm

7 1

0 345

500

1000

410

442 nm 0 10 15 5 [β-CD] × 10-3 M

1

0 375

487.5

Fluorescence intensity (a.u)

Fluorescence intensity (a.u)

7

0 10 15 5 [α-CD] × 10-3 M

492.5

640

Wavelength (nm)

980 If 695

c) pH ~1

50

200

Wavelength (nm) 1000

200 If 125

b) pH ~7

600

950 If 520

d) pH ~7

436 nm 90

0

500

0 345

10 15 5 [β-CD] × 10-3 M

492.5

Wavelength (nm)

640

Wavelength (nm)

Fig. 3. Fluorescence spectra of MFA in different α-CD and β-CD concentrations (M) at pH ~1 and pH ~7: (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.010. Inset Fig.: fluorescence intensity vs. CD concentration. Excitation wavelength ~350 nm.

550

400

500

a) pH ~1

If 450 361 nm 400

0

7

275

10 15 5 [α-CD] × 10-3 M

1

0 290

effect on the absorption spectra. It can be seen in Figs. 3 and 4 that the fluorescence spectral shape of two forms (monocation, neutral) is drastically different in the absence of CDs. The excitation wavelength of MFA was at 355 nm in pH ~ 1 and 335 nm in pH ~ 7, whereas 275 nm for

395

Fluorescence intensity (a.u)

Fluorescence intensity (a.u)

The fluorescence spectra of MFA and ALF in the absence and presence of α-CD and β-CD at pH ~ 1 and pH ~ 7 are shown in Figs. 3 and 4. As is evident from these figures, the effect of CDs on the fluorescence spectra of drug molecules is more pronounced than the corresponding

500

348 nm 300

7 1

0 290 300

450 If 225 0

1 225

361 nm

0 10 15 5 [β-CD] × 10-3 M

7

0 290

395

Wavelength (nm)

5 10 15 [α-CD] × 10-3 M

405

520

Wavelength (nm)

500

Fluorescence intensity (a.u)

Fluorescence intensity (a.u)

c) pH ~1

0

200

Wavelength (nm) 450

400 If 350

b) pH ~7

300 If 285

d) pH ~7 1

70

150

7

348 nm

0

7

10 15 5 [β-CD] × 10-3 M

7 1 1 0 290

405

520

Wavelength (nm)

Fig. 4. Fluorescence spectra of ALF in different α-CD and β-CD concentrations (M) at pH ~1 and pH ~7: (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008 and (7) 0.010. Inset Fig.: fluorescence intensity vs. CD concentration. Excitation wavelength ~280 nm.

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Table 1 Absorption and fluorescence maxima (nm) of MFA in different concentrations of α- and β-CD at pH ~1 and pH ~7. Concentration of CD (M)

pH ~1

pH ~7

α-CD

Water (without CD)

0.004

0.006

0.010

λexcitation (nm) K (M−1) ΔG (kcal·mol−1)

β-CD

α-CD

β-CD

λabs

log ε

λflu

λabs

log ε

λflu

cabs

log ε

λflu

λabs

log ε

λflu

354 280 221 354 279 221 354 279 221 354 281 221

3.68 3.87 4.30 3.69 3.88 4.31 3.73 3.89 4.32 3.75 3.90 4.33

439

354 280 221 354 279 221 354 279 221 355 281 221

3.68 3.88 4.31 3.73 3.90 4.33 3.79 3.93 4.36 3.81 3.96 4.37

439

335 285

3.74 3.97

435 380

335 285

3.74 3.97

435 380

439

339 280 222 348 279 222 352 275 222

3.75 3.98 4.38 3.77 4.00 4.39 3.78 4.01 4.39

437 380

350 280 222 354 277 221 354 276 221

3.76 3.99 4.38 3.79 4.03 4.40 3.82 4.04 4.41

444 379

439

439

440

360 306 −3.44

138 −2.96

441

442

360 336 −3.50

194 −3.17

ALF in both pH solutions. MFA exhibited dual fluorescence band centered at 380 nm (SW) and 435 nm (LW) at pH ~ 7, whereas in pH ~ 1 a structureless broad emission band was observed at 439 nm. An addition of MFA to CD solution caused a remarkable enhancement in drug emission intensities in both pH solutions. Upon increasing the β-CD concentration (at pH ~ 7), the LW emission maximum is red shifted from λflu ~435 nm to 444 nm and the emission intensity is enhanced hugely whereas the SW emission (λflu ~380 nm) intensity is decreased at the same wavelength. Here it is interesting to highlight that like in absorption spectra, with higher β-CD concentrations, the emission maxima and the spectral shape of both pHs (pH ~ 1 and pH ~ 7) are the same which suggests that a similar type of inclusion complexes is formed. In ALF, upon addition of β-CD in pH ~ 7, the LW (λflu ~ 330, 363 nm) fluorescence intensity decreased accompanied by the appearance of new emission at 420 nm, while the SW fluorescence intensity increased. In contrast at pH ~ 1, without appearance of a new longer wavelength band the emission intensity gradually decreased. Further two isoemissive points are also observed in the fluorescence spectra at ~330 and 400 nm. The above results indicate that both drug molecules are entrapped in the CD nanocavities to form stable drug–CD inclusion complexes. However, in the fluorescence spectra, more noticeable changes were obtained in β-CD solutions. This is because, β-CD cavity provides a larger and depth hollow space than α-CD and thus the

440 380 442 380 335 430 −3.65

290 −3.41

443

444

335 535 −3.78

304 −3.44

drug molecules entrapped more deeply, which is an understandable reason for the above spectral changes in both the absorption and fluorescence spectra. Further, both the neutral and anionic species of drugs formed a similar kind of inclusion at higher CD concentrations. In both pH solutions, the presence of isosbestic point in the absorption spectra suggests the formation of 1:1 inclusion complex between the drugs and CD molecules. However, the absorption and emission spectra of drug molecules at pH ~ 1 and pH ~ 7 are similar to each other indicating that a similar type of inclusion complex exists in this system. The association constant (Ka) for the drug–CD inclusion complex has been determined by analyzing the changes in the intensity of the absorption and fluorescence maxima with CD concentrations. The association constant of inclusion complex of drug with CD was determined by using the Benesi–Hildebrand relation [16]. Fig. 5 illustrates the double reciprocal plots for 1/A − A0 and/or 1/I − I0 vs. 1/[CD]. The straight line in Fig. 5 conforms that the stoichiometry of the drug– CD inclusion complex was 1:1. Further the plots of 1/A − A0 and/or 1/ I − I0 vs. 1/[CD]2 give upward curves, which ruled out the possibility for the formation of 1:2 complex. The Ka values were obtained from the slope and the intercept of the linear plots. According to the association constants of all the complexes, β-CD formed more stable complexes than α-CD. The negative ΔG values in Tables 1 and 2 suggest that the inclusion proceeds spontaneously at 303 K.

Table 2 Absorption and fluorescence maxima (nm) of ALF in different concentrations of α- and β-CD at pH ~1 and pH ~7. Concentration of CD (M)

pH ~1

pH ~7

α-CD

β-CD

α-CD

β-CD

λabs

log ε

λflu

λabs

log ε

λflu

λabs

log ε

λflu

λabs

log ε

λflu

Water (without CD)

274

3.97

274

3.97

4.00

4.00

3.99

274

4.00

274

4.02

274

4.03

0.006

274

4.01

274

4.11

274

4.04

348 361 348 361 348 361

274

274

348 362 348 362 347 361

274

0.004

348 362 348 362 348 362

274

4.12

0.010

274

4.03

274

4.17

274

4.06

348 361

274 221

4.17 4.21

λexcitation (nm) K (M−1) ΔG (kcal·mol−1)

267 −3.36

275 281 −3.39

299 −3.43

348, 361 347, 361 318, 347 360, 442 318, 346 360, 444 275 330 −3.48

348 362

275 319 −3.47

327 −3.63

347 361

275 416 −3.50

182 −3.13

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180

1/A-A0

120

0.04

90

0.03

60

0.02

30

0.01

0

0 0

200

400

600

800

1000

1200

0

200

400

150

90

800

1000

1200

60

1000

1200

d) ALF

pH-1 α-CD pH-1 β-CD pH-7 α-CD pH-7 β-CD

0.06

1/I-I0

120

0.09

b) ALF

pH-1 α-CD pH-1 β-CD pH-7 α-CD pH-7 β-CD

600

1/[CD] M-1

1/[CD] M-1

1/A-A0

c) MFA

pH-1 α-CD pH-1 β-CD pH-7 α-CD pH-7 β-CD

0.05

1/I-I0

150

0.06

a) MFA

pH-1 α-CD pH-1 β-CD pH-7 α-CD pH-7 β-CD

0.03 0

30 0

-0.03 0

200

600

400

800

1000

0

1200

200

400

1/[CD] M-1

600

800

1/[CD] M-1

Fig. 5. B–H double reciprocal plots for formation of 1:1 complexes of MFA and ALF with α- and β-CD. (a, b) Plot of 1/A − A0 vs. 1/[CD] and (c, d) plot of 1/I − I0 vs. 1/[CD].

3.2. Effect of solvents In order to understand the micropolarity around the drug molecules, the absorption and fluorescence spectra of MFA and ALF were recorded in various solvents of different polarities and the relevant data are compiled in Table 3. In all the selected solvents, MFA drug provided three absorption bands whereas a structureless absorption band is obtained in ALF. In MFA, the longer wavelength (LW) absorption band comprises the region around 360–335 nm, the middle wavelength (MW) band is around 284 nm and the shorter wavelength (SW) absorption band appears at 222 nm. Among these bands, MW absorption band undergoes a

Table 3 Absorption maxima, fluorescence maxima (nm) and Stokes shift (Δvss, cm−1) of MFA and ALF in selected solvents at 303 K. Solvents

Cyclohexane

1,4-Dioxane

Ethyl acetate

Acetonitrile

2-Propanol

Methanol

Water (pH ~6.5) a

MFA

ALF

λabs

log ε

λflu

Stokes shift

λabs

log ε

λflu

Stokes shift

355 277 222 351 278 221 350 281 221 351 278 222 350 280 220 342 286 216 336 284

Sata

424 374

4568

286

Sata

330 300

4662

3.92 3.88 4.22 3.87 3.97 4.22 4.01 4.09 4.60 4.02 4.11 4.08 3.77 4.01 4.44 3.37 4.05

446 373

6003

281

4.21

310

3329

445 375

6099

280

4.18

7624

434 374

5432

278

4.14

425 401 378 426 401 378 435 379

5042

278

4.06

356 s 346 321 346 328 319 346 329 318 358 345

Sat—saturated.

5912

276

3.99

6773

274

4.00

361 348

7069

7069

slight red shift as an increase in the polarity and hydrogen bond forming capability of the solvents, ascribed to allow π→π⁎ transition of the benzenoid system of MFA. While a significant blue shift is noticed in the LW band simultaneously with an increase in the molar extinction coefficient (log ε). This band can be safely assigned to intramolecular hydrogen bonding (IHB) formed between carboxylic acid and imino groups [17]. This can be confirmed by the evidence obtained from the FT-IR spectrum of the pure drugs. The lowering of O\H stretching frequency at 3042 cm−1 suggests the presence of IHB between the \C_O and NNH groups. Fig. 1 demonstrates the possible intramolecular hydrogen bond between imino hydrogen and carbonyl oxygen atoms of MFA and ALF. Further, the absorption spectrum of MFA in aqueous solution at pH ~ 7.0 is largely blue shifted than that of other solvents, which indicates that the carboxyl group is ionized under this condition. In addition, in all the solvents the absorption spectral characteristics of MFA are slightly red shifted than those of N-phenylanthranilic acid [18] (NPAA: cyc ≈ λabs ~ 360, 286, 219 nm, λflu ~ 425, 326 nm; meth ≈ λabs ~ 348, 289, 218 nm, λflu ~ 430, 390, 310 nm; wat ≈ λabs ~ 330, 285 nm, λflu ~ 430, 390, 290 nm). While a small blue shift was noticed for ALF in comparison to that of diphenylamine (DPA: cyc ≈ λabs ~ 282 nm, λflu ~ 327 nm; meth ≈ λabs ~ 283 nm, λflu ~ 348 nm; wat ≈ λabs ~279 nm, λflu ~363 nm) [19]. The absorption and emission spectra of MFA in aprotic and protic solvents are blue shifted relative to that in nonpolar solvents. This is because the presence of IHB in MFA is

Table 4 Fluorescence decay parameters of MFA and ALF in water (pH ~1) and 0.01 M CD solution. Drugs

8298

MFA

8795

ALF

Medium

Water α-CD β-CD Water α-CD β-CD

Lifetime (ns) τ1

τ2

2.49 2.93 3.97 0.56 0.82 0.98

8.01 10.27 14.30 2.46 1.34 3.59

Pre-exponential factor τ3

a1

a2

3.61 5.07

0.26 0.29 0.35 0.36 0.42 0.51

0.03 0.04 0.05 0.09 0.21 0.26

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bτN

a3

0.07 0.08

3.98 5.32 7.48 1.55 1.79 3.21

N. Rajendiran et al. / Journal of Molecular Liquids xxx (2014) xxx–xxx

affected by highly polar solvents. The above results indicate that the substituent in the phenyl ring is the key factor for the spectral behavior, because of the different electronic densities of the HOMO on each atom. The fluorescence spectra of the above drugs are displayed in Fig. S2. The effect of polarity of the medium on the fluorescence maxima is more pronounced than that of the absorption maxima, which indicates that the excited state properties differ much from those in the ground state. As in absorption, the emission maxima of MFA are slightly red shifted than NPAA, but when compared to DPA no significant shift was noticed in ALF. In all the solvents, both molecules emit dual fluorescence when excited at 335 nm and 275 nm, respectively. It can be seen in Fig. S2 that the drug MFA exhibited decreased fluorescence intensity upon changing the solvent from nonpolar cyclohexane to polar water with significant blue shift in the maxima. The decrease in the intensity of LW emission band with increasing solvent polarity is interpreted as a breakage of the IHB. Thus, only a small blue shifted emission band is observed for changing the solvent from cyclohexane to water, which indicates that IHB is affected in polar (water) solvents. The shift in the fluorescence spectra suggests that the emitting states of the drug molecules are more polar than the ground state. The lower fluorescence intensities of the drug molecules in polar solvents are due to the radiationless decay of the probes, due to the hydrogen bonding. The possible origin of the large Stokes shifted fluorescence emission is already discussed in our earlier results [18–20]. The large Stokes shift indicates

7

strong changes of the geometry in the excited state. The increase in the excited state dipole moment further supported the presence of IHB in the above drug molecules. It is well known that the MFA or ALF presents two conformers; the first one is with IHB (closed conformer I) and the other is without IHB (open conformer II) (Fig. 1) [17]. The data in Table 3, inferred that in polar solvents the IHB strength in MFA is greater than that present in ALF. Also the rupture of the IHB increases as the solvent polarity increases [17]; i.e., in more polar solvents, the intermolecular hydrogen bonding is much more competing with the IHB. In other words, the amount of open conformer II (without IHB) may increase in those of polar solvents like alcohols and water that can possess higher intermolecular hydrogen bond donating ability. In both drug molecules, the existence of principal conformers can be recognized by the rotation of planes that involve the phenyl rings A and B with respect to the plane containing the rest of the molecule around the bonds C(2)\N(7) and N(7)\C(8) in MFA and C(1)\N(12) and N(12)\ C(14) in ALF, respectively. The rotamers of both molecules that involve only B rings are symmetrical. Conformer II that results from the complete rotation of B ring is not symmetrical. However, in both drug molecules, the rotation of A ring implies the rupture of the strong IHB [21] which formed between the \C_O and N NH groups. The experimental and theoretical results indicate that the abovementioned conformers are not totally planar.

2.708 Å 2.580 Å

2.820 Å

2.950 Å 2.657 Å

1.860 Å

c

a

2.959 Å

2.521 Å 2.629 Å

1.905 Å

2.665 Å

2.636 Å 2.558 Å

b

d

Fig. 6. The optimized structures of the most stable 1:1 inclusion complexes of (a) MFA/α-CD, (b) MFA/β-CD, (c) ALF/α-CD and (d) ALF/β-CD at PM3 level of theory.

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N. Rajendiran et al. / Journal of Molecular Liquids xxx (2014) xxx–xxx

Table 5 Energetic features, thermodynamic parameters, HOMO–LUMO energy calculations and physical properties for MFA, ALF, α-CD, β-CD and its 1:1 inclusion complexes obtained by the PM3 method. Properties

MFA

ALF

α-CD

β-CD

MFA/α-CD

MFA/β-CD

ALF/α-CD

ALF/β-CD

E (kcal·mol−1) ΔE (kcal·mol−1) H (kcal·mol−1) ΔH (kcal·mol−1) G (kcal·mol−1) ΔG (kcal·mol−1) S (kcal/mol-Kelvin) ΔS (kcal/mol-Kelvin) EHOMO (eV) ELUMO (eV) EHOMO–ELUMO (eV)

−52.39

−129.54

−1247.62

−1457.75

126.72

49.37

−570.84

−667.55

86.66

−1.96

−676.37

−789.52

−1302.73 −2.72 −446.40 −2.28 −559.07 30.64 0.414 −0.073 −8.88 −0.55 −8.33

−1519.32 −9.18 −548.54 −7.71 −692.35 10.51 0.482 −0.061 −8.69 −0.43 −8.26

−1384.13 −6.97 −526.04 −4.57 −657.44 20.89 0.449 −0.076 −8.88 −0.52 −8.36

−1599.54 −12.25 −629.57 −11.39 −781.87 9.61 0.510 −0.071 −8.86 −0.53 −8.33

0.134 −8.88 −0.33 −8.55

0.172 −8.69 −0.27 −8.42

0.353 −10.37 1.26 −11.63

We have also optimized the ground state geometry of two conformers (with IHB and without IHB) at DFT B3LYP/6-31 + G level of theory [22]. The fully optimized structures of MFA and ALF with and without IHB are shown in Fig. 1. The optimized geometry obviously illustrates an intermolecular hydrogen bonding (IHB) between O(15) and H(22) atoms of MFA. Likewise in ALF, the IHB was observed between O(26) and H(13) atoms. According to the energy calculations, IHB conformer is more stable than that without IHB conformer. The hydrogen bond energy was determined to be 4.24 kcal mol−1 (for MFA) and 6.13 kcal mol−1 (for ALF). This energy was estimated as the change in total molecular energy in the conversation of the optimized structure of conformer I into other conformer II in which the C_O group is rotated about 180° on the bond axis C(1)\C(14) and C(24)\C(25) in former and latter molecules, respectively. The above theoretical investigations revealed that conformer I is stable than conformer II. 3.3. Possible inclusion complexes From the above discussions, the possible inclusion mechanism is proposed as follows: Naturally two different types of inclusion complex formation between drug molecules and CDs are possible: (i) A ring with substitution of carboxylic group is completely captured in the CD cavity; and (ii) B ring (methyl substituted) is captured in the CD cavity. Let us consider the type II arrangement, when the COOH group presents outside of the CD cavity, the spectral shape of the drug molecule should be similar to the aqueous medium (without CD medium). If type I inclusion complex, the COOH group is entrapped within the CD cavity. In this type, the LW absorption maximum of MFA at pH ~ 7 should be marginally red shifted in the CD than in the aqueous medium. But the results in Tables 1 and 2 indicate that the monoanion maxima of MFA are regularly red shifted in α-CD medium. Further in our earlier studies [20,23], we reported that if the \COOH group is entrapped in the CD cavity, the anions in the CD medium are largely red shifted than the aqueous medium. These results support the present case; the association in which the B ring is entrapped completely into the CD cavity is more favored than other types of association. However, the \COOH group may interact with the secondary hydroxyl rim protons of the CD cavity. In 1:1 inclusion complex, the association constants for deprotonated form i.e. monoanion of MFA (pH ~ 7) are considerably higher than those of their neutral form (pH ~ 1). In the case of 1:1 ALF/CD, the greater association constant values are obtained for neutral form of ALF (at pH ~ 1) than deprotonated form (Tables 1 and 2). These results revealed that the association constants are more sensitive to change of pH values in aqueous solution. However, the α-CD complex has lower association constant than that of the β-CD complex. This indicates that the selective association happened between the different molecular forms of drug and CD molecules. Of the two molecular forms, we should note that the β-CD can readily encapsulate the deprotonated species of MFA than the neutral species. This is because CDs are good proton donors [14,24]. The higher association constant in pH ~ 7 implied that the \COO− group is located near the secondary rim of the CD cavity

0.409 −10.35 1.23 −11.58

and interacts with the CD protons, which is further supported by the H NMR results as discussed later.

1

3.4. Time-resolved fluorescence studies The formation of inclusion complex was also analyzed by the fluorescence decay curves obtained in the aqueous, α-CD and β-CD solutions. The fluorescence decay curves of MFA and ALF monitored at 330 nm and 280 nm and the best-fitted parameters are presented in Table 4. The excited singlet state lifetime of MFA is higher than that of ALF in both the environments. In aqueous pH ~ 1 solution, both drugs MFA and ALF exhibit bi-exponential decay indicating the possible existence of two different components with lifetime values τ1 and τ2. The slow decay component probably originated from the locally excited (LE) state. The fast decay component is considered to arise from the IHB state. It can be seen in Table 4 that the relative amplitude for the fast decay component of ALF is lower than that of the slow decay component. A possible explanation would be the deactivation channel of the singlet excited states via intramolecular proton transfer in aqueous solution. It has already been reported by Inoue et al. [25,26] that the organic molecules having ICT properties deactivate efficiency through hydrogen bonding. Therefore, it is likely that the excited molecules in aqueous solution deactivate partially via intermolecular hydrogen bonding, which strongly competes with the TICT state in ALF rather than that in MFA. By the addition of CD (10 × 10− 3 M), biexponential decay curve of ALF drug became triexponential while significant enhancement of the lifetime of MFA is observed. This suggests the existence of new components i.e., complexed form other than the characteristic components of the drug molecules. However, the increase in the fluorescence lifetime of different components of both drugs upon inclusion in the CD is explained from a reduction in the polarity in the vicinity of the fluorophore. With the addition of β-CD, the lifetime of drugs greatly increases in comparison to that of α-CD, owing to the more vibrational restriction in the excited state (S1). Further this is supported by the higher amplitude values obtained for the complexed components in β-CD medium. 3.5. Molecular modeling studies In order to substantiate the information obtained from the experimental results, semiempirical quantum mechanics calculations were carried out at PM3 level of theory [11,12]. This calculation was performed on the structures of the complexes with the lowest energy which were acquired during the simulation process. Fig. 6 displays the geometrical structure of MFA/α-CD, MFA/β-CD, ALF/α-CD and ALF/β-CD complexes with the least energy obtained from PM3 calculations. The energetic features, HOMO energy and LUMO energy and thermodynamic parameters (enthalpy, entropy, free energy) of the guests (MFA and ALF), hosts (α- and β-CD) and its inclusion complexes are summarized in Table 5. According to the energy calculations of the complexes, the orientation in which the B-ring of both guest molecules

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a

9

b

50 µm

c

5 µm

d

e

10 µm

50 µm

g

f

100 µm

20 µm

h

50 µm

10 µm

Fig. 7. SEM photographs of (a) α-CD, (b) β-CD, (c) MFA, (d) MFA/α-CD complex, (e) MFA/β-CD complex, (f) ALF, (g) ALF/α-CD complex and (h) ALF/β-CD complex.

sequestered into the CD cavity is energetically favorable in nature with 1:1 ratio (Fig. 6). It is clear from Fig. 6 that in the complexes, the B-ring of guest molecules is located at the primary rim and the A-ring is positioned nearby the secondary hydroxyl rim. Additionally, it can also be seen that the structure of β-CD complexes is different from that of αCD complexes. In β-CD complexes, both rings (A and B) are almost completely encapsulated within the CD cavity. Nevertheless, the A-ring of the guest molecules remains present outside of the α-CD cavity. The total conformation energy of various complexes was found to be − 1302.73 kcal mol−1 (for MFA/α-CD complex), − 1519.32 kcal mol−1 (for MFA/β-CD complex), − 1384.13 kcal mol−1 (for ALF/α-CD complex) and −1599.54 kcal mol−1 (for ALF/β-CD complex). As is evident from these conformational energy values, the abovementioned orientation is more favorable because these values are lower than the sum of the total energy of guest and host molecules. Further, we also made a careful investigation on the conformational changes of guest molecules that occur during complexation process. Table 4 comprises the assessed geometrical parameters such as bond lengths, bond angles and dihedral angles of free drug molecules and the drug in the CD complexes. The assessed parameters indicated that the geometry of MFA as well as ALF distorted to a great extent after the complex formation. The distortions were significant in dihedral angles. This suggests that the drugs adopted a specific conformation to form a stable inclusion complex. Besides, the conformation of CD is also significantly altered during complex formation (Fig. 6).

The PM3 optimized complex structures in Fig. 6 are also illustrated; there are several intermolecular H-bonds in the structures. Here the H-bonds are defined as O\H⋯O and C\H⋯N with a dH⋯O or dH⋯N distance less than 3.0 Å [27]. From the structures of the four complexes as shown in Fig. 6, we could notice that β-CD formed three H-bonds with the guest molecules and α-CD complex showed two H-bonds. In addition, it is obviously observed that the H-bonding lengths in β-CD complex are shorter than those of α-CD complex. This may explain why the binding energy of the β-CD complex is lower than that of the complex of α-CD. To inspect the thermodynamic parameters of the encapsulation process, the statistical thermodynamic calculations were performed on all the complexes at 1 atm and 298.15 K by the PM3 method. The calculated thermodynamic quantities, the binding energies (ΔE), enthalpy changes (ΔH), Gibbs free energy changes (ΔG) and entropy changes (ΔS) are depicted in Table 5. The binding energy (ΔE) of complexation was calculated for the least energy conformation structures according to Eq. (1). The higher negative value of binding energy corresponds to the more stable complex. In Table 5, among the four inclusion complexes, ALF/β-CD complex (−12.25 kcal·mol−1) has the lowest energy than the other complexes ALF/α-CD (− 6.97 kcal·mol−1), MFA/β-CD (−9.18 kcal·mol−1) and MFA/α-CD (−2.72 kcal·mol−1). The binding energy of inclusion complexes increases as MFA/α-CD b ALF/αCD b MFA/β-CD b ALF/β-CD. This ordering reveals that the stability of the inclusion complex increases depending on the complexation ability of native CD and the nature of the interaction between both the guest

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N. Rajendiran et al. / Journal of Molecular Liquids xxx (2014) xxx–xxx

a

b 48.79 nm

200 nm 200 nm

c

d

200 nm

1 µm

200 nm

1 µm

f

e

200 nm 200 nm

200 nm 200 nm

Fig. 8. TEM photographs of (a, b) MFA/α-CD complex, (c, d) MFA/β-CD complex, (e) ALF/α-CD complex and (f) ALF/β-CD complex.

and host molecules. The above results confirmed that ALF and β-CD formed more stable complex in comparison to that of others. Further, the enthalpy changes (ΔH) and entropy changes (ΔS) are negative (Table 5), suggesting that the formation of the inclusion complexes is an enthalpy-driven process in nature. While the positive free energy changes (ΔG) of the inclusion complexes imply that the inclusion preceded non-spontaneously under the experimental temperature range (298.15 K). Furthermore, the theoretically calculated ΔG values (in kcal mol− 1) were significantly different from the experimental findings: MFA/α-CD, (pH-1) ≈ abs = − 2.96; flu = − 3.44; MFA/β-CD (pH-1) ≈ abs = − 3.17; flu = − 3.50; MFA/α-CD (pH-7) ≈ abs = − 3.41; flu = − 3.64; MFA/β-CD (pH-7) ≈ abs = − 3.42; flu = − 3.78; ALF/α-CD (pH-1) ≈ abs = −3.36; flu = −3.48; ALF/β-CD (pH-1) ≈ abs = −3.47; flu = −3.63; ALF/α-CD (pH-7) ≈ abs = −3.13; flu = −3.39; ALF/β-CD (pH-7) ≈ abs = −3.43; flu = −3.58. Since, the inclusion reaction took place in aqueous solution, the effect of water on the inclusion process should be considered because complexation releases a number of water molecules from the CD cavity. Further, the influence of water molecules

around the guest molecules is very significant. Therefore it is inferred that the effect of water molecules on the complexation process probably changes the encapsulation reaction from a non-spontaneous process in the vacuum phase to a spontaneous process in the aqueous solution. Furthermore, we noticed that the dipole moment values of free guest molecules increased when the guest molecule penetrates into the CD cavity and the complex is formed. We have also investigated the electronic structure of the inclusion complexes in addition to that of isolated drug molecules (Fig. S3). In the HOMO of MFA, the charge density is mainly located on the A ring along with the secondary amino group and small amount of charges spread over the B ring. However in the case of LUMO, more charge density shifts completely to the C\C bond of the phenyl ring (A) and carboxylic acid group. For ALF, the HOMO charge density is positioned on the both rings (A and B) except the alkyl chain and Cl− atoms. Whereas the LUMO charge density is positioned only on the A ring. The HOMO and LUMO energy orbital pictures of drug molecules clearly demonstrated that when the HOMO → LUMO transition takes place an electron density transfers from the amino group to the C\C bond of the

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4. Solid inclusion complex studies 4.1. Scanning electron microscopic morphological observations SEM technique is appropriate to prove the formation of an inclusion complex. It can confirm the existence of one or more components and afford information about particle shape and size. So, first we observed surface morphological structures for pure components by SEM, and then we also observed the surface morphological structure of the inclusion complexes. SEM photographs of MFA, ALF, α-CD, β-CD and their inclusion complexes are displayed in Fig. 7. Pure MFA existed as flake shaped crystal, and ALF was observed as bar-like crystal structures. The micrographs of both CD showed the crystalline nature of the samples. On the contrary in the inclusion complexes, the crystal nature of the drugs disappeared and incidences of hallow amorphous phase material were found which suggest that the drug molecules are well dispersed within the CD nanocavity. These modifications of crystals can be assumed as an evidence for the formation of nano-encapsulated inclusion complexes in solid state. 4.2. TEM analysis Transmission electron microscope (TEM) analysis has been permitted to visualize the different nanostructures directly in this study. Fig. 8 shows typical particle morphologies for samples of MFA and ALF nanoparticles produced in the presence of both α-CD and β-CD. The images in Fig. 8 indicated that MFA/α-CD, ALF/α-CD and ALF/β-CD nanoparticles were predominantly spherical in shape with an irregular surface. The diameter of the nanoparticles based on the TEM micrographs was about 48 nm, 40 nm and 35 nm for MFA/α-CD, ALF/α-CD and ALF/β-CD, respectively. Further the ALF/β-CD nanoparticles were obviously larger than the ALF/α-CD nanoparticles. The reason could be explained that ALF/β-CD inclusion complex formed more easily than ALF/α-CD according to the results of the experimental studies i.e. absorption, fluorescence, time-resolved fluorescence and 1H NMR in solution state. In the case of MFA/β-CD, the well defined spherical structures of micro-aggregates with vesicular shells were observed (Fig. 8c and d). The average diameter of the vesicles is about 720 nm. A mechanism is assumed for the formation of vesicles in the inclusion complex system of β-CD with MFA in aqueous phase as follows.

Exothermic (mW/mg)

(f)

(e) 114.5°C 70.5°C

(d)

103.7°C 124.9°C

150.6°C

40

60

80

100

120

140

160

o

Temperature ( C)

(c)

Exothermic (mW/mg)

phenyl ring and carboxylic group. It was also found that the charge densities on each atom of the drug molecule within the CD cavities were slightly altered. In general, the energy gap between EHOMO and ELUMO is an important scale of stability [28], and the molecules with larger energy gap tend to have higher stability. The calculated EHOMO–ELUMO results are presented in Table 5. In our case, both drug–β-CD complexes have lower energy gap suggesting that β-CD formed more stable complexes with drug molecules as compared to α-CD. The lowering of the energy gap is fundamentally a result of the larger stabilization of LUMO due to the strong electron-acceptor ability of the electronacceptor group. We also noticed that the energy gap of PFO/α-CD complex just falls in the range than that of the drug molecule, suggesting that there will be no significant change in the electronic spectra of the guest molecules upon the binding and molecular encapsulation, which agrees well with the experimentally determined results in pH ~ 1 solutions (neutral form). The energy gap also explains the eventual charge transfer interaction with the inclusion complexes, which influences the biological activity of the drug–CD inclusion complexes. It is expected that stronger charge transfer interaction would take place in ALF/CD complexes than MFA/ CD complexes, as the HOMO of ALF lies significantly higher than that of MFA. But while looking at Table 5, the HOMO energy of all the complexes is similar to each other except MFA/β-CD where larger HOMO energy was observed. This indicates a parallel type of charge transfer interactions between the drug molecules and CDs.

11

(b) 108 °C 106.5 °C

(a)

75.3 °C

131 °C

231.5 °C

50

100

150

200

250

Temperature (oC) Fig. 9. DSC thermograms of (a) MFA, (b) MFA/α-CD, (c), MFA/β-CD (d) ALF, (e) ALF/α-CD and (f) ALF/β-CD in 1:1 molar ratio (25–260 °C at 10 °C/min).

As mentioned earlier the drug molecule formed inclusion complex with β-CD in a 1:1 molar ratio at much lower concentration. In the 1:1 MFA/β-CD inclusion complexes, the carboxylic acid functions of several drug molecules could form strong hydrogen bonds with each other and thereby enhance the stability of the supramolecular complex for the formation of vesicles in the presence of water. Numerous water molecules are highly assisted for the formation these vesicles. For verification of the importance of water molecules in vesicle formation, a small amount of alcohol was added to the same samples. After the addition of alcohol, the stability of produced vesicles decreased and thus the well defined surface collapsed. These observations are similar to those obtained by Lo Meo and coworkers [29] for β-CD with p-nitroaniline derivatives. Further Lizhen Sun and coworkers [30] demonstrated that the 1:2 stoichiometry between N,N′-bis(ferrocenyl methylene) diaminohexane and carboxymethyl β-cyclodextrin (CMβ-CD) turned into 1:1 molar ratio by the addition of alcohols to the water. They also observed vesicles with relatively weak stability in the water/ethanol mixture (1:2 v/v) than those obtained in water. 4.3. FT-IR spectral analysis The possible interaction between the drug molecules and hydrophobic CD nanocavities in solid state was investigated by FTIR spectroscopy. Fig. S4 illustrated the FT-IR spectra of MFA, ALF and its corresponding solid inclusion complexes. The IR spectrum of pure MFA is presented in Fig. S4a. It was found that the band at 3312 cm−1 for NH stretching vibration is consistent with the IR spectrum corresponding to the MFA form-I

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N. Rajendiran et al. / Journal of Molecular Liquids xxx (2014) xxx–xxx

10.25

12.25

26.30

f 21.15

12.10

25.75

e 25.6 17.02

22.03

d 10.8

the NH stretching vibration at 3317 cm−1 completely disappeared in the complex, while the intensity of C_C ring stretching (1582 cm−1 and 1506 cm−1) decreased and shifted to 1588 cm−1 and 1509 cm−1. Further the C\Cl stretching vibrations at 749 cm−1 and 664 cm−1 both shifted in the inclusion complex (753 cm−1 and 667 cm−1). This is because the aromatic ring with Cl entrapped into the CD cavities. The IR spectral analysis of the above pure drugs (i.e. MFA and ALF) and its solid inclusion complexes revealed considerable changes in the IR peaks of the drug molecules when complexed with excipients and it confirmed the presence of strong interactions between the drugs and CD nanocavities through inclusion complex formation.

13.65

22.67

c 12.20 14.13 16.7

22.3

b

4.4. 1H NMR spectral studies The formation of inclusion complexes can be confirmed from the changes observed in the chemical shifts of MFA/ALF or CD in 1H NMR spectra [15]. Figs. S5 and S6 display 1H NMR spectra for the free drugs and its inclusion complexes with α-CD and β-CD. In these figures, the spectral assignations of the guest molecules are shown as insets. The chemical shift values for the MFA, ALF, and α-CD/β-CD inclusion complexes (in parentheses) are as follows:

Fig. 10. Powder XRD patterns of (a) MFA, (b) MFA/α-CD, (c) MFA/β-CD, (d) ALF, (e) ALF/α-CD and (f) ALF/β-CD.

MFA: Ha = 12.985 (13.001/13.011), Hb = 9.441 (9.474/9.472), Hc = 7.866 (7.864/7.869), Hd = 7.289 (7.305/7.319), He = 7.115 (7.190/7.202), Hf = 7.106 (7.125/7.129), Hg = 7.017 (7.037/7.042), Hj = 6.697 (6.706/6.705), Hk = 6.669 (6.684/6.687), Hl = 2.274 (2.290/2.292), Hm = 2.104 (2.089/2.090); ALF: Ha = 13.129 (13.127/13.128), Hb = 7.524 (7.509/7.506), Hc = 7.215 (7.210/ 7.208), Hd = 7.059 (7.058/7.058), He = 6.953 (6.941/6.933), Hf = 6.854 (6.859/6.850), Hg = 4.633 (4.622/4.619), Hj = 6.254 (6.242/ 6.239), Hk = 3.892 (3.881/3.880).

reported by Gilpin and Zhou [31] and Romero et al. [32]. The band at 3312 cm−1 arises from the amino group internally hydrogen bonding with the carbonyl group (MFA form-I). Besides, other bands were observed at 1649 cm−1 for C_O stretching vibration and 1257 cm−1 for C\N stretching vibration. The strong C_C stretching vibrations of the aromatic ring appeared at 1575 cm−1, 1510 cm−1, 1482 cm−1 and 1450 cm−1 and the four weak bands for the overtones and combinations are observed in the region 2000–1700 cm−1. Further the bands at 1193 cm−1, 1161 cm−1, 1095 cm−1, 1047 cm−1, 1039 cm−1 and 754 cm−1 are obtained for CH in-plane bending and CH out-of-plane bending vibrations of the aromatic ring. From the FTIR spectra of pure drug and complexation of drug with CDs, it was observed that all the characteristic peaks of MFA are absent in the spectra of solid inclusion complexes indicating that the drug encapsulated within the CD cavity. The NH stretching vibration, the overtones and combination bands for the aromatic ring and the weak band for CH3 deformation (in the region 1400–1360 cm−1) completely disappeared in both α- and β-CD inclusion complexes. This suggests that the methyl substituted aromatic ring along with the secondary amine (NH) group may be included in the CD cavity, though the strong band for C_O stretching (1649 cm−1) was shifted to 1652 cm−1 in the inclusion complexes. In Fig. S4d, the prominent IR absorption peaks of ALF are showed at 3317 cm−1, 1770 cm− 1 and 1718 cm−1 corresponding to NH, ester and C_O stretching, respectively. As in pure MFA, the broad band at 3317 cm−1 may arise from the intramolecular hydrogen bonded amino group with the carbonyl group (ALF form-I) [33]. The CH stretching superimposed on OH stretching vibrations appeared at 2935 cm−1. Further the band at 749 cm−1 and 664 cm−1 is obtained for C\Cl stretching vibrations and the C_C aromatic ring stretching observed at 1582 cm−1 and 1506 cm−1. It can be seen from the spectrum of Figs. S4e and 5f that there were obvious changes in the IR spectra of pure drug after the inclusion complex ALF/CD was formed. For example,

It can be seen in Figs. S5 and S6 that most of the aromatic protons of the MFA are hugely influenced owing to the presence of CD. Shielding of chemical shift values is observed for all the aromatic protons of both A and B rings as well as for amino protons (Hb). However, the major induced upfield shifts are observed for all the B-ring protons in both drug/α-CD and drug/β-CD complexes. Additionally in MFA, the methyl protons (Hl) showed outstanding upfield chemical shifts in comparison to the Hm protons in the inclusion complex. The significant difference for these 1H NMR spectra strongly confirmed the solid inclusion complex formation. From the above discussions we can conclude that MFA drug was included into hydrophobic nanocavity of CD. The 1H NMR spectrum of ALF in DMSO-d6 consists of nine types of protons. In the presence of CD, the chemical shifts value of ALF is remain approximately constant. It is observed from the 1H NMR spectra of inclusion complexes that strong downfield shifts in the resonances for Hb, He, Hg, Hj, and Hk protons indicate the interaction between ALF and CD. In fact, the proton signals corresponding to the phenyl ring-B are shifted significantly in comparison to those of the phenyl ring-A, which suggests that this phenyl moiety (B) is involved in the complex formation. This deduction is in well agreement with the experimental results obtained in aqueous solution. To further investigate the inclusion manner, the chemical shifts of CD protons in the absence and in the presence of drug molecules should be considered. By comparing the chemical shifts of these protons after inclusion complex formation with ALF, a comparably weak shift was observed on the δ values of the outer side protons of CD (H-1, H-2, H-4 and H-6). In contrast, those values of H-3 and H-5 protons which are located inside of the CD cavity exhibited the significant changes (~0.07 and 0.06 ppm). It is also noteworthy that H-3 protons of α-CD shifted largely (~0.09 ppm), but after the complex formation H-5 protons showed relatively weak shifts (~0.04 ppm). These phenomena suggested that ALF enters more deeply into the β-CD cavity from the secondary face rather than α-CD.

6.2

15.7

21.2

26.3

a

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4.5. DSC analysis The thermal curves of pure MFA and ALF, pure α-CD and β-CD and the corresponding equimolar solid complexes are depicted in Fig. 9. The DSC curves of pure drugs were typical of anhydrous crystalline substances, exhibiting initially a flat profile followed by a sharp melting endotherm at about 231.5 °C (MFA) and 150.6 °C (ALF), respectively. The DSC profile of α-CD showed three endothermic peaks at ~ 79 °C, 109 °C and 137 °C and that was related to dehydration of water molecules from the cavities. However, β-CD exhibited a wide range of endothermic band between 70 °C and 140 °C, which attains a maximum at 128.6 °C, attributed to that of dehydration process. On the contrary, the thermal curves of solid products of both drugs with CD showed a remarkable decrease in intensity of the CD endothermic peak in comparison with pure material, as well as a simultaneous shift at lower temperature. Further, MFA and ALF melting peak completely disappeared in the α-CD and β-CD inclusion complexes. All the above results are indicative of strong drug–CD interactions in the inclusion complexes and the drugs are well dispersed in the CD nanocavities [34,35]. 4.6. XRD analysis The X-ray diffraction (XRD) patterns of MFA, ALF, α-CD, β-CD and their corresponding solid inclusion complexes are presented in Fig. 10. It was found that the formation of inclusion complex alters the original crystal lattice of CD and consequently the X-ray diffraction pattern. The XRD patterns of both the pure drugs indicated high crystallinity due to the characteristic sharp peaks at diffraction angle 2θ values of 6.2°, 13.7°, 14.9°, 15.7°, 20.0°, 21.2°, 26.3°, 27.6°, 31.3°, and 40.4° (for MFA) and 8.5°, 11.2°, 14.2°, 17.3°, 18.4°, 19.2°, 22.0°, 24.4°, 25.6°, and 31.9° (for ALF). Both α-CD and β-CD displayed typical crystalline diffraction patterns. As observed by many researchers [34,35], for the physical mixture, only a slight decrease in peak intensity was noticed. However, the patterns showed several peaks attributable to both crystalline drugs and CD and it is highly possible to distinguish the characteristic peaks of host and of guest molecules. On the contrary, the diffraction pattern of solid inclusion complexes exhibited low crystallinity with numerous weaker intensity peaks and some amorphous curves, in which the diffraction peaks of the drug and CD disappeared. Moreover, the diffraction pattern of the peaks of the inclusion complexes was highly diffused as compared to raw materials (MFA/ALF and CD). Comparative analysis of these diffractograms evidently proved that the true inclusion complex formed between the above drugs and CD molecules. These observations were in agreement with the results of the DSC analysis.

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driving force for the inclusion process and also responsible for the complex stability. The statistical thermodynamic calculations suggested that these inclusion complex processes are enthalpically favorable in nature. Acknowledgments This work is supported by the CSIR [No. 01(2549)/12/EMR-II] and UGC [F. No. 41-351/2012 (SR)] New Delhi, India. 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 facilities. The authors thank Dr. P. Ramamurthy, Director, National centre for ultrafast processes, Madras University for the help in the fluorescence lifetime measurements for this work. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.molliq.2013.11.027. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]

5. Conclusions

[24]

The inclusion complexes of MFA and ALF with α-CD and β-CD were studied by absorption, fluorescence, time-resolved fluorescence and molecular modeling methods. Absorption and fluorescence spectral data indicate that phenyl ring-B (methyl substituted) of both molecules was present in the inner part of the CD nanocavity and phenyl ring-A (COOH substituted) was present in the hydrophilic part. The timeresolved fluorescence studies also confirmed the formation of inclusion complex. SEM, FT-IR, 1H NMR, DSC and XRD results suggest that the drugs formed solid inclusion complexes with hydrophobic nanocavities of CD. The inclusion complexes of drugs–CD self-assembled into nanoparticles except MFA/β-CD for which vesicles were observed by TEM. Semiempirical calculations confirm that the hydrogen bonds were the

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