Synthetic clay mineral as nanocarrier of sulfamethoxazole and trimethoprim

Applied Clay Science 161 (2018) 395–403

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Applied Clay Science journal homepage: www.elsevier.com/locate/clay

Research paper

Synthetic clay mineral as nanocarrier of sulfamethoxazole and trimethoprim a

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D. Hernández , L. Lazo , L. Valdés , L.C. de Ménorval , Z. Rozynek , A. Rivera

a,⁎

T

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Zeolites Engineering Laboratory, Institute of Materials Science and Technology (IMRE), University of Havana, Cuba Department of Pharmacy, Institute of Pharmacy and Food (IFAL), University of Havana, Cuba Institut Charles Gerhardt Montpellier, Equipe Agrégats, Interface, et Matériaux pour l'Energie (AIME), Université Montpellier 2, France d Faculty of Physics, Adam Mickiewicz University, Poland b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Sulfamethoxazole Trimethoprim Antibiotics Li-fluorohectorite Synthetic clay mineral

In the present work the incorporation of two model drugs, sulfamethoxazole (SMX) and trimethoprim (TMP) on the Li-fluorohectorite (LiFHt), synthetic clay mineral was evaluated. Understanding the interactions established between these two antibiotics – which are complementary forms from the pharmaceutical point of view – and LiFHt, allows to develop new drug delivery formulations with both drugs in the same support. The quantification of both drugs was followed by ultraviolet (UV) spectroscopy. For the interaction clay mineral-drugs, different physical-chemical parameters−pH, drug initial concentration, temperature and interaction time−were studied. The results showed that, for both drugs, the best clay mineral-drug nanocomposites were obtained at acid pH, room temperature during 1 h, and initial drug concentration of 3 mg/mL. The resulting clay mineral-drug nanocomposites were characterized by X-ray diffraction (XRD), infrared (IR) spectroscopy and thermal gravimetry (TG). It was corroborated by XRD that the TMP was truly intercalated into the clay mineral. However, SMX seems to be adsorbed onto the clay mineral surface. For the LiFHt-TMP nanocomposite, IR suggested clay mineral-drug interactions via amine groups of the TMP. No significant changes in the IR spectrum for the LiFHtSMX were observed. The drug release profiles showed to follow the pharmaceutical standards, and suggested the possibility to design formulations of drug delivery using LiFHt as carrier.

1. Introduction It is well known that conventional release dosage forms provide an immediate drugs release, without much control of the release rate. In order to obtain therapeutically effective plasmatic concentrations, and to avoid significant fluctuations in the plasmatic drug levels, it is necessary to achieve dosage control. Failing to do so can lead to drug levels in the organism, by excess or defect, resulting in undesirable side effects, or in the lack of therapeutic benefits for the patient. Such disadvantages can be reverted through the use of materials to control the drugs release (Siegel and Rathbone, 2012; de Sousa et al., 2013). Several reports about the use of clays and clay minerals in the pharmaceutical industry like active principles and/or excipients can be found (Aguzzi et al., 2007; de Sousa et al., 2013). Among the many benefits offered by clays, is their safety for the human health. The desirable physical and chemical properties of clay minerals−as adsorbents and ion exchangers−make them play a substantial role in pharmaceutical formulations (Aguzzi et al., 2007). For example, clays from the smectite family have been used as support materials in drug slow release systems. However, the use of synthetic clay minerals offers

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the possibility to optimize the conditions for the incorporation of model drugs, because this way the interference of spurious phases in the interpretation of the results is avoided. The Li-fluorohectorite, that belongs to the smectite group, is a 2:1 clay mineral with a negative charge net where a fraction of Mg2+ ions are substituted by Li+ in trioctahedral sites resulting in a negative structural charge of −1.2 electrons per unit cell (Kaviratna et al., 1996). It is compensated by exchangeable hydrated cations, i.e., Li located between clay layers allowing their stacking. The stacks can swell in the presence of water, which may enter the interlayer space, increasing the distance between layers. Based on the swelling property of this clay mineral, a few studies about its use as support system for pharmaceutical applications have been reported (Rivera et al., 2016; Valdés et al., 2016, 2017a, 2017b; dos Santos et al., 2017). Trimethoprim (TMP) and sulfamethoxazole (SMX) are complementary pharmaceutical forms. The synergy between both drugs was first described in a series of in vitro and in vivo experiments published in the late 1960s (Maddileti et al., 2015). TMP or 2,4-diamino-5-(3,4,5trimethoxybenzyl)pyrimidine is a broad spectrum, synthetic antibacterial agent, which acts as an inhibitor of bacterial dihydrofolate

Corresponding author. E-mail address: [email protected] (A. Rivera).

https://doi.org/10.1016/j.clay.2018.03.016 Received 14 November 2017; Received in revised form 6 March 2018; Accepted 9 March 2018 Available online 26 May 2018 0169-1317/ © 2018 Elsevier B.V. All rights reserved.

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To evaluate the influence of pH on the incorporation process of TMP and SMX on the LiFHt, acid, neutral and basic pH values were considered. Taking into account the pKa values of both drugs, the pH studied for TMP were around 3.5, 7 and 12. For the case of SMX, four pH values (about 1, 3.5, 7 and 12) were evaluated. The pH was carefully adjusted using concentrated solutions of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The drug amount in the supernatant solutions were determined by means of UV spectroscopy relative to calibration curves for the pure drugs solutions, taking into account the absorption maxima: for TMP, λmax = 271 nm at acid pH, λmax = 285 nm at neutral pH and λmax = 288 nm at basic pH. For SMX, λmax = 265 nm at acid pH and λmax = 256 nm at basic pH. The dispersions were stirred during 2 h at room temperature at 3 mg/mL of initial drug concentration. To evaluate the effect of the initial concentration for TMP and SMX, the interactions were performed in the range of 0.5–3 mg/ mL–considering its solubility in aqueous medium (Li et al., 2005)–and 1–9 mg/mL (50 mg/100 mL at pH 8.54 and 1550 mg/100 mL at pH 5.5 (Dahlan et al., 1987)), respectively. The study was made at pH about 3.5 for 2 h and room temperature. The influence of temperature was also studied, performing the experiments at 27, 45 and 65 ± 1 °C, at pH about 3.5 for 2 h and 3 mg/mL of initial drug concentration. For kinetics studies the interactions were carried out during 30 min, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h at pH around 3.5, room temperature, initial drug concentration of 3 mg/mL and constant stirring. All the experiments were replicated three times in order to demonstrate their repeatability. The maximum difference between the outputs and their average was of 10 mg, which corresponds to a relative uncertainty of around of 3% in the mass of incorporated drug.

reductase, belonging to a group of compounds known as diaminopyrimidines (ElShaer et al., 2012). SMX or 4-amino-N-(5-methyl-3-isoxazolyl) benzenesulfonamide, is a bacteriostatic antibiotic belonging to the sulfonamides family. It is effective against most gram-negative and gram-positive bacteria and is frequently used for the treatment of urinary infections. Sulfonamides are structurally analogous to the natural substrate and competitive inhibitors of para-aminobenzoic acid (PABA) (Goodman-Gilman et al., 1991). The aim of this study is to evaluate the potential of synthetic Lifluorohectorite for the incorporation and release of the model drugs TMP and SMX and to understand the interactions between both materials using different characterization techniques (XRD, IR and TG). An in vitro drug release study from the clay mineral-drug nanocomposite tablets, simulating gastrointestinal tract conditions, was also performed. The main motivation of the study of these drugs is to develop new clay mineral-drug nanocomposite materials based on the combination of both drugs in the same support (LiFHt) with potential applications as a delivery system. 2. Experimental 2.1. Materials The LiFHt used in the experiments was purchased from Corning Inc., New York. It contains about 80% by mass of LiFHt with nominal formula Lix(Mg6−xLix)F4Si8O20 with x = 1.2, and about 20% of Li2O·2SiO2 impurities (Rivera et al., 2016). In vivo acute toxicity assays performed on the LiFHt in Wistar albino rats indicated no clinical signs of toxicity in the organs, when the animal groups under study were examined (Valdés et al., 2017b). Based on that, it was possible to conclude that LiFHt can be used as raw material for medical applications, according to standard pharmaceutical requirements. Thus, it was demonstrated that the Li+ amount present in the clay mineral-drug nanocomposite obtained does not represent any health problem. Trimethoprim (C14H18N4O3) and sulfamethoxazole (C10H11N3O3S)—pharmaceutical grade according to the United States Pharmacopoeia (USP30-NF25, 2007)—were the model drugs studied. They were used as received from the Cuban pharmaceutical industry. All other chemicals used in the study were analytical grade.

2.4. Structural, spectroscopic and thermal characterization The solid samples in powder form before and after the interaction were analyzed with a Philips Xpert diffractometer, using Cu Kα radiation (λ = 1.54 Å) at room temperature, operating at a voltage of 45 kV and working current 25 mA. The angular scanning range 2θ from 2° to 40°at a scan rate of 1° min−1. Fourier transform infrared spectra (FTIR) were collected using a Nicolet AVATAR 330 Fourier–transform IR spectrometer in the wavenumber interval 400–4000 cm−1. The samples were prepared using KBr pellet with 0.8% inclusion of the material to be analyzed. Thermal gravimetric analysis (TGA) was performed with the aid of a NETZSCH STA 409 PC/PG and a STA6000 Perkin Elmer thermal analyzer, using a heating rate of 10 °C/min under dry air flow, from 25 up to 500 °C. The sensitivity of the thermobalance was ± 1 μg. A solid sample of about 0.03 g was used in each test.

2.2. Sample preparation For the interactions, the general procedure followed was: 10 mL of drug aqueous solutions was put in contact with 100 mg of LiFHt powder. After that, the mixture was centrifuged for 15 min at 300 rpm. The clay mineral-drug nanocomposites were dried at 65 °C. The drugs concentrations after the clay-drug interaction were analyzed and quantified by ultraviolet (UV) spectroscopy, using a spectrophotometer Rayleigh UV-2601 in the wavelength interval 200–400 nm. The drugs uptake by the synthetic clay mineral (i.e., adsorbent loading) was calculated as:

qe =

2.5. Drug release assays For the preliminary release studies different media, synthetic gastric juice (SGJ, i.e., 2 g of NaCl in 1 L of HCl 0.1 N), synthetic intestinal juice (SIJ, i.e., 6.8 g of K2HPO4, 0.2 L of NaOH 0.2 N and add water up to 1 L) and synthetic combined juice (SCJ, i.e., a mixture of SGJ and SIJ adjusting the pH to 4.5) were used, in order to simulate the conditions along the gastrointestinal tract (GIT). The sequential release was conducted in a buffer solution at pH 1.2 (synthetic gastric juice without pepsin) for 2 h (gastric emptying time). After that, the dissolution media was replaced with a buffer solution of pH 7.4 (synthetic intestinal juice) and analyzed for further 6 h. The assays were carried out with the resulting materials in tablet form with about 30 mg of the clay mineral-drug nanocomposite (equivalent to 6.5 mg of TMP or 5.6 mg of SMX determined by UV as described before) and a compression agent in a Relation 1:1. The microcrystalline cellulose is a direct compressing agent accepted by the United States Pharmacopoeia (USP) for its use pharmaceutical formulations. The tablets were placed in dialysis membrane bags (Spectra/

(Co − Cf ) × V m

(1)

where qe (mg/g) is the mass of drug adsorbed per unit mass of the adsorbent, Co is the initial drug concentration in solution (mg/mL), Cf is the final concentration of drug solution (mg/mL), V is the volume of solution (mL) and m is the mass of the adsorbent (g) used in the experiments. 2.3. Drug incorporation and pre-formulation of the drug carrier system In order to determine the best conditions to increase the drug load per gram of clay mineral, the influence of several physical-chemical parameters (pH, initial drug concentration, temperature and time of contact) was studied. 396

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7.3 (Liu et al., 2012), the TMP is in its divalent cationic form due to the protonation of the two amine groups. It would facilitate the interaction with the clay mineral surface negatively charged due to clay-drug electrostatic interactions. However, when comparing the TMP load at acid and basic pH, the difference was not significant, which indicates that these interactions are not crucial in the formation of the clay mineral-drug nanocomposite. For SMX, the drug adsorption took place basically at acid pH, and an important decrease of the SMX amount adsorbed on the clay mineral at neutral and basic pH was observed (Fig. 1b). At pH about 1, the hydrogen ions concentration is very high, establishing a competition between them and the drug molecules by the active sites of the clay mineral, which leads to a decrease of the SMX amount adsorbed on the clay mineral. At neutral pH, the SMX is negatively charged. Thus, repulsive interactions drug-clay are expected, i.e., the SMX amount on the clay mineral decreases. At basic pH, the amount of SMX in milligram per gram of material was very small, which suggests a low affinity of the drug for the clay mineral. The best results, expressed as milligram of SMX per gram of LiFHt, were obtained at pH about 3.5, reaching efficiency in the incorporation process around 83%. In such case, the most important contribution to the incorporation process occurred when the drug is in its neutral form. Therefore, the experimental data suggested that the interaction between both edrug and claye occurs through weak interactions, i.e., Van der Waals forces and/or hydrophobic interactions.

Por 4 Dialysis Membrane, 30 m × 32 mm, MWCO 12–14 kD) of approximate dimensions 2 × 4 cm2, and they were immersed in 50 mL of dissolution medium at 37 °C, and constant stirring at 100 rpm, according to the procedure reported in the Pharmacopoeia (USP30-NF25, 2007). Aliquots of 2 mL of the solution were taken at regular time intervals and the same volume was replaced with a fresh dissolution medium in order to simulate the passage through the GIT. The procedure was performed by triplicate for each the clay mineral-drug nanocomposite sample. The average values of release percentage were reported, with uncertainty around 6%, which corresponds to maximum difference in release percentage between the average and the replicas. To evaluate the mechanism of drugs release from the clay mineraldrug nanocomposite materials, various mathematical models were used to describe the release kinetics. The selection of best fit was based on comparison of the determination coefficient (r2). 3. Results and discussion 3.1. Effect of the chemical and physical parameters on the drugs incorporation on the LiFHt

3.1.2. Effect of initial drug concentration The dependence between the initial drug concentration in solution and the efficiency of drug adsorption process on LiFHt are shown in Fig. 2. As can be seen in the inset, for both drugs, the load per gram of clay mineral increased linearly with the initial drug concentration. However, the efficiency of the drug incorporation process had a different behavior: for TMP, the efficiency was around 95% for all concentration values studied. In the case of SMX, the efficiency increased with the drug concentration up to 5 mg/mL, reaching a maximum value of about 90%, from which it does not vary significantly. This behavior is expected, assuming that the incorporation processes are mainly diffusive. It is based on the establishment of a concentration gradient between the liquid and solid phases (i.e., from the phase with the highest concentration of drug to the lowest concentration). Considering the process efficiency (i.e., percentage of drug adsorbed on the clay mineral

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3.1.1. Influence of the drug-clay mineral dispersion pH As shown in Fig. 1, the pH effect in the TMP and SMX incorporation on LiFHt. The results suggested that the TMP adsorption is favored at acid pH (about 93% efficiency of the process, see Fig. 1a). It can be explained based on the pKa value of TMP: at pH below of a pKa around

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Fig. 2. Efficiency of the drug incorporation process as a function of the initial drug concentration of solution. The efficiency has been calculated as the percent of drug incorporated on the clay mineral relative to the initial amount of drug in the solution. The inset shows the load of incorporated drug as a function of the initial drug concentration.

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pH Fig. 1. TMP incorporation (a) and SMX (b) on the LiFHt as a function of pH. 397

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Table 1 Dependency of the sulfametoxazole (SMX) and trimethoprin (TMP) incorporation on the LiFHt with the temperature. 27 °C

45 °C

65 °C

SMX TMP

250 ± 10 mg/g 280 ± 10 mg/g

235 ± 10 mg/g 271 ± 10 mg/g

211 ± 10 mg/g 272 ± 10 mg/g

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Fig. 4. Diffraction patterns for LiFHt, LiFHt-TMP and LiFHt-SMX.

each one of the models drugs. From the practical point of view, 1 h as time interaction was selected for both drugs.

3.2. Samples characterization Considering the result above discussed, the best clay mineral-drug nanocomposites (LiFHt-SMX and LiFHt-TMP) were obtained under the following conditions: initial drug concentration of 3 mg/mL, a pH about 3.5, contact time of 1 h, and room temperature (27 °C). The amount of drug in milligram incorporated per gram of clay mineral (load) was 250 ± 10 mg per g of clay mineral for the LiFHt-SMX nanocomposite and 280 ± 10 mg per g of clay mineral for the LiFHt-TMP nanocomposite material. From the analytical point of view, the SMX amount per gram of clay mineral could be estimated as follows: Mx(Mg6−xLix)Si8O20F4 is the chemical formula of the unit cell of LiFHt (where M = Li representing the exchangeable cations and x ≈ 1.2), which has an atomic mass of about 754. The atomic mass of the SMX molecule is about 254, which allows an estimate of the SMX to clay mineral mass ratio of around 0.34 for a compactly packed clay mineral sample (it has assumed that approximately 1 drug molecule is associated to one unit cell of the LiFHt as a lower limit of the fully loaded clay mineral, as in (Rivera et al., 2016)). Considering that the LiFHt sample under study typically has 40% mesoporosity (Knudsen et al., 2003), the estimated of SMX load is approximately 0.2. From previous work (Rivera et al., 2016), the X-ray diffraction and atomic adsorption spectrometry studies suggested that around 80% of the mass of the powder samples was clay mineral, and that the remaining 20% were impurities. Assuming that the impurities do not play a role in the capture of drug, the SMX mass incorporated per unit mass of clay mineral would be 0.24. This value is in total agreement with that obtained in the present study, where the experimental results by UV spectroscopy indicated that the SMX mass incorporated per unit mass of sample is 0.25. A similar analysis was performed for the TMP, where both the estimation and experiment resulted in 0.28 for the TMP mass incorporated per unit mass of sample. FTIR spectra of the clay mineral-drug nanocomposites (LiFHt-TMP and LiFHt-SMX) and the starting materials (LiFHt, TMP and SMX) are given in Fig. 3. In the LiFHt spectrum can be identified the characteristic bands associated with the normal SieO and HeO vibration modes. The bands at 3448 and 1643 cm−1 were associated to the stretching vibration OeH (νOeH) and bending in the plane (δH2O) corresponding to the water molecules in the interlayer space, respectively. The antisymmetric and symmetric stretching SieO of the tetrahedral layer (νSieOas and νSieOs) at 1091 and 997 cm−1, respectively, were ascribed.

Fig. 3. Infrared transmittance spectra for samples of (a) SMX, LiFHt, and LiFHtSMX; (b) TMP, LiFHt and LiFHt-TMP. The characteristics bands are indicated for each one.

relative to the drug mass in the initial solution before interaction with clay), the initial drug concentration selected was 3 mg/mL for both drugs.

3.1.3. Effect of temperature, and interaction time In both cases, the drug mass on LiFHt did not evidence a significant dependence with the temperature (Table 1). For SMX, the amount per gram of clay decreased slightly with the temperature increase, which in terms of process efficiency does not imply important differences. The results suggested that the interactions established between the model drug and the active sites of the clay were weakened with the temperature increase. Thus, all further experiments were conducted at room temperature (27 °C). Regarding time, the amount of drug incorporated at different time intervals (from 30 min to 24 h) revealed that the adsorption incorporation process was very fast. The amounts SMX and TMP in milligram per gram of LiFHt was very similar during all the time range for 398

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Fig. 5. Chemical structure and dimensions of the molecules of TMP (a) and SMX (b).

Fig. 6. Schematic representation of the load of TMP and SMX on the LiFHt.

The out-of-plane SieO bending band at 710 cm−1 and the in-plane bending at 472 cm−1 were also observed (Farmer, 1958; Molu and Yurdakoç, 2010; Rivera et al., 2016). The strong absorption bands observed in the TMP spectrum―drug in neutral form―at 3471 and 3319 cm−1 correspond to the asymmetric and symmetric NeH stretching vibrations of the amino group (νNH2as and νNH2s, respectively). The characteristic bands that appear in the 3250 to 2800 cm−1 region were assigned to the CeH stretch of the pyrimidine ring, the benzyl aromatic and methyl groups: the first is a broad band centered at 3124 cm−1 due to Csp2eH vibration of valence (νCsp2eH), the other two at 2929 and 2831 cm−1 are due to Csp3eH stretching (νCsp3eH) (Maddileti et al., 2015). The band at 1633 cm−1

was attributed to the bending of the amino group (δNH2s). The signals at 1597, 1564 and 1463 cm−1 were assigned to the C]N stretching of pyrimidine ring and C]C aromatic ring stretching. The stretching frequencies around 1236 and 1126 cm−1 were ascribed to CeOeC vibrations (νCeOeCasand νCeOeCs) (Burbuliene et al., 2009; Garnero et al., 2010; ElShaer et al., 2012; Macedo et al., 2012; Maddileti et al., 2015). In the LiFHt-TMP spectrum (see Fig. 3b), the presence of typical bands of TMP on the clay mineral was recognized. In the region of 3800–2800 cm−1 the drug bands appear overlapped with the broad absorption band of the adsorbed water in the clay mineral. The interaction process between the LiFHt and the TMP drug takes place at acid

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Fig. 7. TG and DTG curves for (a) TMP, LiFHt and LiFHt-TMP; and (b) SMX, LiFHt and LiFHt-SMX.

corresponding to the 001 basal reflection, at 2θ = 7.3°. The d-value, calculated according to Bragg's Law for this angle was 1.21 nm. This value agrees with that reported in the literature for fluorohectorites with a water layer in the interlayer space (Silva and Fossum, 2002; Hemmen et al., 2010). After the TMP-LiFHt interaction a significant spacing of clay mineral layers was observed in the pattern (see Fig. 4): an intense and broad reflection at 2θ = 4.1°, with a d value of 2.15 nm was observed, confirming the true intercalation of TMP in the interlayer space of the LiFHt. However, in the LiFHt-SMX diffractogram, the incorporation of the SMX in the clay mineral did not produce a significant variation of the interplanar distance (2θ = 7.2° corresponding to d = 1.23 nm). This result suggested that SMX is not intercalated in the LiFHt interlayer space, suggesting its adsorption on the clay mineral surface. The shift in the inter-planar distance corresponding to the characteristic reflection of the LiFHt (001 basal reflection), due to the presence of the TMP and SMX drugs was of 0.94 and 0.02 nm, respectively. Such difference could be explained considering the functional groups and the degree of ionization as a function of pH (net charge), in each case: for example, for the SMX, from the molecular dimensions point of view―around 0.89 nm × 0.80 nm × 0.72 nm (see Fig. 5)―, it would be expected at least its partial incorporation into the interlayer space. However, if functional groups of the molecule are considered, and that the best adsorption of SMX is obtained at pH around 3.5 (SMX in its neutral form), its incorporation in the interlayer space is not obvious. Maybe, the explanation could be based on the highest affinity between the SMX molecules, and in their interaction with an “open” surface (clay mineral surface), without the confinement impediment that may take place in the interlayer space. On the other hand, in the case of TMP even though it has larger dimensions (0.81 nm × 0.92 nm × 1.12 nm, Fig. 5) than SMX, after the interaction with the LiFHt an increment in the d-value from 1.21 to 2.15 nm takes place. It could be understood considering that the best incorporation is found when the functional groups of the TMP molecule are positively charged, i.e., clay mineral-drug interaction at acid pH. A schematic representation of the atomic structure of LiFHt, and a sketch of a possible configuration of LiFHt with the TMP intercalated

pH (i.e., the drug is in cationic form). The band corresponding to NeH bending of the NH2 group (δNH2s) of the drug, appears at 1670 cm−1 (a shift to higher vibration frequencies, 37 cm−1, relative to TMP alone). It is known that the absorption of protonated amino groups (eNH2+) in the infrared region occurs at lower frequencies due a weakening of the bond (Heacock and Marion, 1956). However, in the IR spectrum of the LiFHt-TMP, an increment towards higher frequencies in the band corresponding to NeH bending of the NH2 group was observed. The drug in cationic form (TMP2+) will interact with the clay mineral negatively charged via interlayer intercalation and/or surface adsorption. It indicates a strong interaction between the positively charged amino groups of TMP and the clay mineral. The characteristic bands of the SMX, drug in neutral form by absence of charge (see Fig. 3a) observed at 3467 and 3377 cm−1 were assigned to the antisymmetric and symmetric stretching of the primary amine group (νNH2as and νNH2s). The band at 3296 cm−1 was assigned to the stretching of the sulfonamide group (νNeH). The band at 3142 cm−1 corresponds to the CeH valence vibration (νCsp2eH) of the aromatic ring and the heterocycle. The bands near to 1621 cm−1 can be assigned to the NeH bending of the NH2 group (δNH2s), and to the combination of the phenyl group (νC]C) and izoxazole ring (νCeN) vibrations. The bands at 1311 and 1154 cm−1 were attributed to the antisymmetric and symmetric stretching of the sulfonyl group, respectively (Kesimli, 2001; Kesimli and Topacli, 2003; Braschi et al., 2010; Refat et al., 2010; Mondelli et al., 2013). LiFHt-SMX spectra (Fig. 3a) displayed characteristic bands of the SMX, which confirms its presence in the clay mineral-drug nanocomposite. In its spectrum, the typical bands of the drug appear overlapped with the clay mineral bands, particularly in the regions of 3677–3030, 1729–1543 and 1239–903 cm−1. No significant shifts in the characteristics bands of the starting materials were observed, confirming a weak clay-drug interaction through Van der Waals forces and/or hydrophobic interactions. The diffraction patterns for LiFHt and clay mineral-drug nanocomposites (LiFHt-TMP and LiFHt-SMX) are shown in Fig. 4. The XRD pattern of the LiFHt indicated the presence of only one reflection, 400

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90 80

TMP release (%)

and the SMX adsorbed on the clay mineral surface is shown in Fig. 6. TG/DTG curves for the starting materials (TMP, SMX and LiFHt) and the clay mineral-drug nanocomposites (LiFHt-TMP and LiFHt-SMX) are shown in Fig. 7. Two prominent peaks of mass loss, around 100 °C and 155 °C, can be identified in the DTG curve for the clay. These peaks could be attributed to water desorption, in particular to the loss of mesoporous and intercalated water, respectively (Rivera et al., 2016). At higher temperatures the dehydroxylation of the LiFHt took place, resulting in the observation of a decomposition peak at 530 °C in the DTG curve (Bergaya et al., 2006; Rozynek et al., 2012). In the TG and DTG diagrams of TMP (see Fig. 7a) it is evidenced that the thermal decomposition starts at 270 °C up to 375 °C, in a single step. This result is in agreement with that reported in the literature for this drug (Macedo et al., 2012). For the SMX (Fig. 7b), the decomposition took place in two steps: a first mass loss between 180 and 380 °C, and a second mass loss from 470 to 750 °C. In the DTG curve of the drug, three peaks can be identified: a doublet at 258 and 273 °C, related to thermal decomposition of the drug, and the other two peaks, at 310 and 630 °C, attributed to the final thermolysis of SMX (Rivera and Farías, 2005). In the TG/DTG diagrams for the LiFHt-TMP, the peaks corresponding to the clay mineral water loss, appear at lower temperatures compared to the raw material. It could be due to the TMP intercalation in the interlayer space (Rivera et al., 2016). The peaks around 277 °C and 350 °C in the DTG curve can be attributed to the decomposition of TMP in the clay mineral-drug nanocomposite. The comparison between the DTG curve of the TMP alone and that of the LiFHt-TMP nanocomposite suggested a significant variation in the decomposition process of the drug (i.e., the decomposition occurs at smaller temperatures after the clay mineral interaction). It could indicate that the TMP thermal stability decreases in the clay mineral-drug nanocomposite. In the TG/DTG curves of the LiFHt-SMX nanocomposite a three-step decomposition was observed. In the first step two peaks at 80 and 150 °C, corresponding to the clay mineral water loss, were identified. In particular, the loss of mesoporous water occurred at lower temperatures in the clay mineral-drug nanocomposite material regarding LiFHt. It suggests a weak interaction between these water and the clay mineral surface, due to their displacement by the SMX molecules adsorbed in the clay mineral-drug nanocomposite. Thus, such result matches with those obtained by XRD, where it was verified that the SMX is on the clay mineral surface. The other two decomposition steps were due to the SMX presence in the clay mineral-drug nanocomposite. In the DTG curve it can be observed four peaks at 256, 292, 370 and 520 °C. Comparing the DTG curve of the LiFHt-SMX nanocomposite and the SMX alone, variations in the final drug thermolysis were evidenced. These shifts occurred at lower drug decomposition temperatures indicating lower stability of SMX in the clay mineral-drug nanocomposite.

SGJ pH=1.2 SCJ pH=4.5 SIJ pH=7.4

70 60 50 40 30 20 10 0 0

1

2

3

4

5

6

Time (h) 100

SMX Release (%)

90

SIJ pH = 7.4 SGJ pH = 1.2 SCJ pH = 4.5

(b)

80 70 60 50 40 30 20 10 0 0

1

2

3

Time (h)

4

5

6

Fig. 8. In vitro release profile of (a) TMP from LiFHt-TMP nanocomposite and (b) SMX from a LiFHt-SMX nanocomposite in synthetic gastric juice (SGJ), synthetic combined juice (SCJ) and synthetic intestinal juice (SIJ) at 37 °C. 110 100

Drugs Release (%)

90

SMX

SGJ pH= 1.2

80

3.3. In vitro drug release

70

The release profiles of TMP and SMX from the LiFHt-TMP and LiFHt-SMX nanocomposites at different pH at 37 °C over 6 h are shown in Fig. 8 (a and b). The amount of TMP released depends on the pH medium and the percent of drug released increased at higher pH values. At low pH values (i.e., pH < 1.78), the TMP dissolution would be dominated by the diffusion of protons, whereas at higher pH values the total flux would become dependent upon all species present in the (TMP, H+, OH−, etc.) (Dahlan et al., 1987). As can be seen in Fig. 8a, for the different media, the load of TMP released from the LiFHt-TMP nanocomposite over 2 h were 15, 30 and 50% in SGJ, SCJ and SIJ, respectively. In the SMX case, it was also observed a strong pH dependence. The SMX amount released was strongly related with the SMX solubility at the different pH media. It increases significantly with both the increase and the decrease of pH due to the formation of the drug salts (for example, about 182 mg/100 mL at pH 1.15 (Dahlan et al., 1987)). In SCJ (pH = 4.5) at 2 h, the amount of SMX released was

60

TMP

50 40 30

SIJ pH= 7.4

20 10 0 0

1

2

3

4

5

6

7

8

Time (h) Fig. 9. Sequential release profiles of TMP and SMX from LiFHt-TMP and LiFHtSMX nanocomposites, respectively.

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contact time. In such conditions, the drug loads on the clay mineral were 250 mg of SMX and 280 mg of TMP per gram of LiFHt. The structural characterization by XRD allowed to verify the true intercalation of TMP into the interlayer space, while the SMX was adsorbed on the clay mineral surface. In vitro release studies of drugs from the LiFHt-SMX and LiFHt-TMP nanocomposites showed that the clay mineral promotes the controlled release of SMX and TMP, and that the percentage of drugs released increases both with time and with pH of medium.

Table 2 Fit of sequential release profiles of TMP and SMX at different mathematical models. Mathematical model

Equation

TMP r2(%)

SMX r2(%)

Zero order First order Higuchi

C = C0 − kt ln(C) = ln(C0) − kt

96.6 88.7 96.0

99.2 88.7 98.1

94.7 (n = 0,87)

99.8 (n = 0,87)

96.6

98.8

Korsmeyer and Peppas Hixson-Crowell

1 Mt = kt 2 M∞ Mt = ktn M∞ 3 Ccd = kt

Acknowledgments

C: drug concentration dissolved in time, Co: initial drug concentration, k: rate constant, Mt/M∞: fraction of drug released at time t, n: release exponent, Ccd: drug concentration. no dissolved in time.

The authors thank the Organizing Committee of the 16th International Clay Conference and University of Havana (Travel Grant VRI 2017) for the financial support. Professor J. O. Fossum is acknowledged for providing the raw clay mineral and facilitating the inclusion of the work in the Scientific Research Abstracts. The authors thank Professor E. Altshuler for the discussions and critical revision of the manuscript. A. Rivera thanks The Academy of Sciences for the Developing World (TWAS) for research Grants No. 00-360 RG/CHE/LA and No. 07-016 RG/CHE/LA. Z. Rozynek acknowledges financial support of the Polish National Science Centre through OPUS programme (grant number 2015/19/B/ST3/03055). Laboratorios MEDSOL are acknowledged for providing the pharmaceutical grade raw materials.

around 16% of the amount of initial drug present in the LiFHt-SMX nanocomposite. In SGJ and SIJ, for the same time, the SMX amount released was 40 and 74%, respectively (Fig. 8b). In comparison with commercial forms existing in the market, under similar release conditions, the clay mineral-drug nanocomposites obtained can maintain the drugs for a longer time: for example, the release profiles of TMP and SMX from commercial or “more standard” formulations show that nearly 80–100% of the drugs are released around 30 min (Lin and Kawashima, 1987; Athanassiou et al., 1991; Phothipanyakun et al., 2013; Medina et al., 2015). In conclusion, as the clay mineral-drug interactions are favored, the amount of drug released will be smaller. The sequential release profile was obtained (Fig. 9), where the pH of the solution was changed from SGJ to SIJ, in order to simulate the transit of the clay mineral-drug nanocomposite along the gastrointestinal tract. During the first 2 h, the percentage of drug released was very similar to the separate experiments shown in Fig. 8a and b. After that, the medium was switched to SIJ and a gradual increase of the drug released (70% for TMP and 90% for SMX) was observed. In general, the profiles for each drug, SMX and TMP, show clearly the different interactions SMX-clay mineral relative to the TMP-clay mineral. The highest SMX percentages released with respect to TMP suggests a stronger interaction between the last drug and the LiFHt. It is rightly supported by the infrared spectroscopy results for the clay mineral-drug nanocomposites, where a shift of the TMP characteristics bands is observed when it is compared with the TMP spectrum alone. The high complexity of the physical-chemical phenomena involved in the process of releasing the drug from the clay mineral-drug nanocomposite casts doubts on the existence of a universal release mechanism valid for all systems. Different mathematical models (Table 2) were used to describe the mechanism of drug release in the sequential release profiles. The best fit for SMX release was found for Korsmeyer and Peppas model with release exponent n = 0.87. This result indicated that the SMX release from the clay mineral-drug nanocomposite took place through the combination of diffusion and erosion processes. In the case of TMP release, Zero order and Hixson-Crowell models were the ones that best describe the data behavior with similar determination coefficients (r2 = 96.6%).

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