1. Introduction
Oral administration is the main route of drug administration for a systemic effect [
1]. Orally administered drugs should be released from the dosage form and dissolve before absorption. Thus, numerous attempts such as complexation, particle size reduction, solid state alternation, the application of soft gel technology, solid dispersions, using cosolvents or forming emulsions, microemulsions, micelles, polymeric micelles, liposomes, pharmaceutical salts, and pro-drugs have been made to increase the dissolution rate of the drugs in order to improve their bioavailability [
2]. Particle size reduction is an easy and suitable approach to increase the dissolution rate and thus the absorption due to the increment in specific surface area [
3]; however, the stabilization of particle size may be a critical issue in this approach.
Solid state dispersion is a particularly promising technique to enhance the solubility, dissolution rate, and bioavailability of poorly water-soluble drugs and to resolve the stability problems of micronized/nanonized drugs, which are dispersed in an inert solid carrier or matrix either as fine particles or molecularly [
4]. This technique has numerous advantages from many aspects, such as improved stability due to the probable interactions between the drug and carrier functional groups [
5], the increment of glass transition temperature of the solid dispersion matrix [
6] or the displacement of crystalline structure by an amorphous form [
7,
8], resulting in local solubility and wettability improvement of poorly soluble drugs [
9], and suppression of drug precipitation from the supersaturated solution to achieve higher solubility and dissolution rate for the metastable drug polymorphs connected to the carrier [
10]. Solid dispersions may be divided into multiple classes including solid solutions, drug–carrier complexes, glassy solutions or suspensions, simple eutectic mixtures, and amorphous drug precipitates in a crystalline carrier [
11]. Solid state dispersions can be prepared with various methods including the fusion process, solvent method, fusion-solvent method, spray drying, lyophilization, hot-melt extrusion, the electrospinning method, supercritical fluid technology, and spraying on beads using a fluidized-bed coating system [
2]. In the solvent method, the drug and the carrier are dissolved in a suitable solvent, which will later be evaporated at an elevated temperature or under vacuum. Then, supersaturation and simultaneous precipitation of the components happens, resulting in a solid residue. Afterwards, organic and/or toxic solvents should be completely removed under vacuum. For this purpose, many sensitive techniques can be used to detect the trace amounts of solvents, such as differential scanning calorimetry (DSC), thermogravimetric (TG) analysis, or differential thermal analysis (DTA) [
2]. The upsides of this method are the ability to control drug particle size by monitoring the temperature and the solvent evaporation rate [
3], the capability of evaporating solvents at a lower temperature, and reduced pressure for thermolabile drugs or for frozen systems [
2]. The downsides of this method are the difficulty of choosing the appropriate solvent for both the drug and the carrier, since most of the carriers are hydrophilic while the drugs are hydrophobic [
12], the necessity of complete solvent removal, especially if the solvents can plasticize the carrier [
13], and the large volume of solvent required to dissolve both the drug and the carrier, which is not economical in some cases [
2].
Conventional drugs have many limitations, such as restricted drug solubility, undesirable pharmacodynamics, side effects, short circulating time, and lack of selectivity [
14,
15,
16,
17]. Of the drugs currently on the market, 90% are hydrophobic and poorly soluble or insoluble in water, which restricts systemic delivery [
18]. However, nanocarriers may improve solubility, absorption, permeation, and retention in the target tissues, as well as the bioavailability, circulation time, and stability of drug molecules [
19]. Furthermore, they may protect various drug molecules from premature degradation in the body and show higher uptake efficiency in the target cells compared to normal cells [
20]. Nanotubes not only have an exemplary inner diameter of 5–6 nm, which makes them able to contain therapeutic drugs and large biological molecules, but also a large surface area and distinct outside geometry, which enable them to be modified and multi-functionalized [
21].
Titanate nanotubes (TNTs) have particularly appealing characteristics, such as hydrophilicity, biocompatibility, high surface area, stable tubular structures [
22], controllable dimensions, tunable geometries, surface chemistry, and the ability to modulate drug release kinetics [
23]. In addition, layered titanate nanostructures have been used in several industrial applications, such as pharmaceutics, energy storage, photocatalysis, electronics, paints, and coatings. Moreover, 1D titanate nanomaterials are receiving more scientific interest, evidenced by the fact that about one new paper is published daily, according to an ISI Web of Science topic search [
23]. Furthermore, TNTs can be used as drug carriers since they can load a higher amount of drug compared to carbone nanotubes (CNTs) [
24].
In a previous work, 70% ethanol solution was used as a solvent to prepare composites with various drugs [
25]. The selected solvent was able to improve the poor aqueous solubility of diclofenac sodium, atenolol (ATN), and hydrochlorothiazide (HCT) [
26,
27,
28] or improve the crystallization of the highly water-soluble diltiazem HCl, while providing uniform process conditions for better comparability. Nevertheless, the result revealed that the composite formation was suboptimal for ATN and HCT. According to our hypothesis, the suboptimal solubility of the drug in the solvent, the featured crystal growth due to slow evaporation, and the intensive drug–solvent interactions may be possible explanations. The present study aims to optimize the composite formation of TNTs with atenolol (ATN) and hydrochlorothiazide (HCT), thus improving the solubility and bioavailability of the active pharmaceutical ingredients (APIs). These drugs have poor bioavailability due to different reasons. ATN belongs to the 3rd class of the Biopharmaceutical Classification System (BCS), so it has good solubility but poor permeability, which results in a lower than 50% absorption rate from the GI tract, especially if it is taken with food. HCT belongs to BCS class IV, so poor bioavailability is due to poor solubility and permeability. The successful binding of these drugs to an appropriate nanocarrier in nanocrystalline or especially in amorphous form may considerably increase their bioavailability. Therefore, the selection of the optimal solvents and process conditions is essential for such drugs.
2. Materials and Methods
The titanate nanotubes (TNTs), TNT-ATN (TiATN), and TNT-HCT (TiHCT) composites were prepared at the University of Szeged’s Department of Applied and Environmental Chemistry following the general composite formation method described by Sipos et al. [
25]. However, since composite formation was not completely successful in the previous study, the 70% ethanol solution was replaced with methanol (0.0168% water content) and 0.01 M aqueous solution of HCl (HCl 0.01 M) or with 1 M aqueous solution of sodium hydroxide (NaOH 1M), DMF (0.012% water content), and DMSO (0.027% water content) for the synthesis of TiATN and TiHCT, respectively. The water content of the solvents was determined with Karl–Fisher titration. The solvents were purchased from Molar Chemicals Ltd., Budapest, Hungary. In addition, since TNTs exhibit instability below pH 2, TNTs treated with HCl 0.01 M were prepared as a reference to detect if this solvent may cause any change in the properties of titanate nanotubes.
ATN and HCT were kindly supplied by TEVA Pharmaceuticals PLC, Debrecen, Hungary and Gedeon Richter PLC, Budapest, Hungary, respectively. The excipients used for tablets were Avicel PH 112 (FMC Biopolymer Inc., Philadelphia, PA, USA), Tablettose 70 (Meggle Pharma GmbH, Wasserburg am Inn, Germany), talc, and magnesium stearate (both from Molar Chemicals Ltd., Budapest, Hungary).
Hydrothermally synthetized TNTs were prepared by adding 120 g of NaOH in 300 mL of distilled water on a magnetic stirrer for a few minutes and then adding 75 g of TiO2 for 15 min. After that, the mixture was put in the autoclave at 185 °C for 24 h then cooled at room temperature for 2 h, followed by cooling with cold water. TNTs were washed with distilled water under vacuum and by using filter No:4.
TNTs with HCl 0.01 M were prepared by adding 50 g of TNTs in 300 mL of HCl 0.01 M in an ultrasonic bath until a homogenous suspension was obtained. After that, 200 mL of HCl 0.01 M was added to the previous suspension on a magnetic stirrer and the mixture was dried in a dry oven for 24 h to remove the solvent.
A 1:1 ratio of TiATN–methanol and TiATN–HCl composites were prepared by adding 50 g of TNTs in 300 mL of methanol in an ultrasonic bath until a homogenous suspension was obtained and 50 g of atenolol in 200 mL of methanol on a magnetic stirrer. After that, the two mixtures were added to each other on the magnetic stirrer, and the final mixture was put in a vacuum distillation device until complete removal of the solvent.
To prepare a 1:1 ratio of TiHCT–NaOH composite, 50 g of TNTs were put in 1000 mL of NaOH 1 M in an ultrasonic bath to get a homogenous suspension, and 50 g of HCT was dissolved in 500 mL of NaOH 1 M on a magnetic stirrer until complete dissolution. Furthermore, the two prepared mixtures were added to each other on a magnetic stirrer until reaching homogeneity. After that, 130 mL of HCl 37% was added to neutralize the final mixture, which was washed with distilled water in a vacuum dryer until pH = 9 to eliminate the solvent. Finally, the obtained powder was dried in a dry oven for 24 h to get the required composites TiATN and TiHCT.
A 1:1 ratio of TiHCT–DMF and TiHCT–DMSO composites were prepared by adding 50 g of TNTs in 1000 mL of DMF in an ultrasonic bath and 50 g of HCT with 1000 mL of DMF on a magnetic stirrer. Then, the two prepared mixtures were added to each other on a magnetic stirrer until a homogenous mixture was obtained, which was put in a vacuum distillation device to remove the solvent.
The morphology and size of the TNTs and composites were investigated by scanning electron microscope (SEM) (Hitachi 4700, Hitachi Ltd., Tokyo, Japan) and transmission electron microscope (TEM) (FEI Tecnai G2 20 X-TWIN, Hillsboro, OR, USA). The APIs, TNTs, and the composites were coated with a thin conductive gold layer by a sputter coating unit (Polaron E5100, VG Microtech, London, UK) for the SEM measurements. The images were taken at an accelerating voltage of 10.0 kV, the used air pressure was 1.3–13 mPa during the analyses. TEM images were taken at 100 kV of electron energy, and those images served to analyze the particle size of TNTs by using Image J 1.47 t (National Institute of Health, Bethesda, MD, USA) software.
To detect the interactions between the APIs and the TNTs, a Thermo Nicolet Avatar 330 FT-IR spectrometer (Thermo Fisher Scientific Ltd., Waltham, MA, USA) was used. Measurements were performed with a Transmission E.S.P. accessory by using 256 scans at a resolution of 4 nm and applying H2O and CO2 corrections. Results were evaluated with Spectragryph 1.2.8 software (Friedrich Menges, Obersdorf, Germany). For better comparability of the original spectra of ATN and HCT with the TiATN and TiHCT composites, respectively, the signal of TNTs was subtracted from the composite spectra and the spectra were normalized to the highest peak which belongs to C=O stretching.
The surface free energy of the prepared samples was determined with a DataPhysics OCA20 (DataPhysics Instruments GmbH, Filderstadt, Germany) optical contact angle tester by using the sessile drop method. Polar and apolar test liquid (water and diiodomethane) were used and dropped onto the surface of 13-mm-diameter tablets prepared with a Specac hydraulic press (Specac Ltd., Orpington, UK) at a pressure of 3 tons. Disperse (γ
sD) and polar (γ
sP) components of the total surface free energy (γ
s) of the solid were calculated according to Wu Equations (1) and (2).
where γ
1 is the surface tension of the first and γ
2 is the surface tension of the second liquid.
Polarity was calculated according to the following Equation (3):
The thermal behavior of TNTs, APIs, and composites was determined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis. TGA and DSC tests were performed by a Mettler Toledo TGA/DSC1 simultaneous analyzer (Mettler-Toledo Ltd., Budapest, Hungary) in which the samples were heated steadily from 25 to 500 °C with a heating rate of 10 K/min, using nitrogen as purge gas. The mass of the samples was 10 ± 1 mg in a closed aluminum pan (100 µL). The curves were evaluated with STARe Software (Mettler-Toledo Ltd, Budapest Hungary). To compare the curves of the API, TNTs, and the composite, the results were normalized to sample weight and to the temperature of the reference pan.
Tablets containing APIs or API–TNT composites (
Table 1) were formulated to study drug release. The powders were mixed with a Turbula mixer (Willy A. Bachofen Maschinenfabrik AG, Muttenz, Switzerland) for 8 min without magnesium stearate and for an additional 2 min with it. Tablets (300 mg) containing 50 mg of API were prepared with a Korsch EK0 eccentric tablet press (E. Korsch Maschinenfabrik GmbH, Berlin, Germany) instrumented with strain gauges and a displacement transducer using 10-mm-diameter flat punches and a 5 kN compression force for all compositions.
Drug release was determined with an Erweka DT700 (Erweka GmbH, Heusenstamm, Germany) dissolution tester using the USP II method. Dissolution was applied at 37 °C using pH 1.2 enzyme-free artificial gastric juice as dissolution media. Samples of 5 mL were taken after 5 min, 10 min, 15 min, 30 min, 60 min, 90 min, and 120 min. The concentration of the released drug was measured with a ThermoScientific GENESYS 10S UV–Vis spectrophotometer (Thermo Fisher Scientific Ltd., Waltham, MA, USA), and the results were evaluated with Sigmaplot v12 (Systat Sofware Inc., San Jose, CA, USA) software.
4. Discussion
Based on these results, it may be concluded that choosing the appropriate solvent is essential from the aspect of composite formation efficacy. The solubility of the API in the selected solvent is important as regards process performance and economy, but its volatility and protic/aprotic nature seems to be more important from the aspect of the strength and quality of composites. ATN exhibits good solubility in 70 w/w% ethanol solution, methanol, and HCl 0.01 M. The Janus-faced properties of the TiATN–ethanol sample may be explained by the fast supersaturation of the solution as a result of the fast evaporation of the ethanol content followed by the slower removal speed of water, which induced the concentration of ATN molecules and featured the formation of ATN–ATN bonds instead of ATN–TNT ones. This latter effect was not observed during the fast removal of water-free methanol, while in the case of 0.01 M HCl solution, the repulsive effect between protonated ATN molecules prohibited the formation of ATN–ATN interactions despite the slower solvent removal speed. The featured drug–carrier interactions and the stabilization of the drug in the nanocrystalline state highly improved the dissolution rate from the TiATN composites.
In contrast, the results with HCT were not so obvious. HCT was dissolved in 70 w/w% ethanol solution and NaOH 1 M as an analog of the experiments with ATN. However, the quality of the TiHCT–NaOH composites was below expectations, which may be explained by the strong interaction between the drug and the solvent, which may lead to the deprotonation of the drug molecule and may decrease the intensity of drug–carrier interactions within the composites. To eliminate this effect, a pair of aprotic solvents was applied, and DMF and DMSO were successful in forming the TiHCT composites since H-bonding was featured between HCT and TNTs due to the lack of drug–solvent interactions. Nevertheless, it is notable that despite the stabilized nanocrystalline form or HCT “recrystallizing” from DMSO in an amorphous form, the very strong drug–carrier interactions resulted in an extended release of the drug from the composites.
In conclusion, the selection of a highly volatile aprotic solvent may be the best way for the preparation of strong TNT–API composites, but the use of protic solvents could also be advantageous if it results in the protonation of the drug molecule. Nevertheless, care must be taken regarding the strength of drug–carrier interactions since they may influence the detachment of the drug and therefore exhibit considerable influence on the characteristics of the products, determining processability, release rate, and behavior in a biological environment.