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Stabilizing Unstable Amorphous Menthol through Inclusion in Mesoporous Silica Hosts Teresa Cordeiro,† Carmem Castiñeira,‡ Davide Mendes,† Florence Danède,§ Joaõ Sotomayor,† Isabel M. Fonseca,† Marco Gomes da Silva,† Alexandre Paiva,† Susana Barreiros,† M. Margarida Cardoso,† Maria T. Viciosa,∥ Natália T. Correia,§ and Madalena Dionisio*,†

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LAQV-REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal ‡ Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, Butanta, 05508-000 São Paulo, Brasil § Univ. Lille, CNRS, UMR 8207, UMET, Unité Matériaux et Transformations, F-59000 Lille, France ∥ CQFM−Centro de Química-Física Molecular and IN−Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, University of Lisbon, Avenida Rovisco Pais, 1049-001 Lisbon, Portugal ABSTRACT: The amorphization of the readily crystallizable therapeutic ingredient and food additive, menthol, was successfully achieved by inclusion of neat menthol in mesoporous silica matrixes of 3.2 and 5.9 nm size pores. Menthol amorphization was confirmed by the calorimetric detection of a glass transition. The respective glass transition temperature, Tg = −54.3 °C, is in good agreement with the one predicted by the composition dependence of the Tg values determined for menthol:flurbiprofen therapeutic deep eutectic solvents (THEDESs). Nonisothermal crystallization was never observed for neat menthol loaded into silica hosts, which can indicate that menthol rests as a full amorphous/supercooled material inside the pores of the silica matrixes. Menthol mobility was probed by dielectric relaxation spectroscopy, which allowed to identify two relaxation processes in both pore sizes: a faster one associated with mobility of neat-like menthol molecules (α-process), and a slower, dominant one due to the hindered mobility of menthol molecules adsorbed at the inner pore walls (S-process). The fraction of molecular population governing the α-process is greater in the higher (5.9 nm) pore size matrix, although in both cases the S-process is more intense than the αprocess. A dielectric glass transition temperature was estimated for each α (Tg,dielc(α)) and S (Tg,dielc(S)) molecular population from the temperature dependence of the relaxation times to 100 s. While Tg,dielc(α) agrees better with the value obtained from the linearization of the Fox equation assuming ideal behavior of the menthol:flurbiprofen THEDES, Tg,dielc(S) is close to the value determined by calorimetry for the silica composites due to a dominance of the adsorbed population inside the pores. Nevertheless, the greater fraction of more mobile bulk-like molecules in the 5.9 nm pore size matrix seems to determine the faster drug release at initial times relative to the 3.2 nm composite. However, the latter inhibits crystallization inside pores since its dimensions are inferior to menthol critical size for nucleation. This points to a suitability of these composites as drug delivery systems in which the drug release profile can be controlled by tuning the host pore size. KEYWORDS: amorphous state, menthol, flurbiprofen, molecular mobility, THEDES, mesoporous silica matrixes, drug release

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INTRODUCTION Water solubility and gastrointestinal permeability are key parameters involved in the extent of drug absorption and bioavailability.1 The latter is a fundamental property for pharmaceutical drugs since the higher the bioavailability, the lower the required dose to achieve the desired therapeutic effect, minimizing the number of unwanted side effects2 and environmental impact.3 Despite efforts made in drug design, about 40% of the existing drugs in the market and about 75% of developing drugs have high hydrophobicity and, as a result, poor bioavailability.4 Therefore, raising the dissolution rate of poorly soluble drugs maintaining their pharmacodynamics is a noteworthy challenge © 2017 American Chemical Society

to the pharmaceutical industry. Preparation as an amorphous material is a strategy adopted by formulation scientists to improve the solubility of poorly water-soluble, crystalline drugs.5 Such is the case of menthol, the target compound in the present work, which in addition to exhibiting therapeutic activity6 is also used as a food7 and cosmetics additive.8 Indeed, the amorphization of pharmacologically active materials is drawing a lot of attention. The amorphous form Received: Revised: Accepted: Published: 3164

May 9, 2017 July 26, 2017 August 2, 2017 August 24, 2017 DOI: 10.1021/acs.molpharmaceut.7b00386 Mol. Pharmaceutics 2017, 14, 3164−3177

Article

Molecular Pharmaceutics

In this work, menthol was loaded into MCM-41 and SBA-15 silica matrixes with pore diameters of 3.2 or 5.9 nm, aiming to access the menthol amorphous state and respective mobility. The composites menthol/silica were studied by DRS, differential scanning calorimetry (DSC), powder X-ray diffraction (XRD), and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The guest release was analyzed by gas chromatograph/mass spectrometry (GC/MS) in full (FM) and single monitorization mode (SIM). Taking advantage of the behavior of menthol in THEDES form, the THEDES approach was used by combining menthol with flurbiprofen in order to estimate the glass transition temperature of neat menthol, which, to the best of our knowledge, is unknown. Finally, a correlation between the molecular dynamics and the delivery profile of menthol loaded into porous silica matrixes is proposed.

corresponds to a disordered arrangement of molecules lacking a distinguishable crystal lattice, which enhances solubility and dissolution rate9 and can potentiate bioavailability10 and therapeutic activity.11 In addition, amorphization is a means to overcome the formation of a polymorphic form or conversion between polymorphic forms. These can exhibit completely different physicochemical properties, as in the case of the polymorphs of menthol.12 However, since the amorphous form is a high energy state with excess entropy, enthalpy, and free Gibbs energy, it is thermodynamically unstable, and thus a strategy is needed to overcome this instability. Several approaches have been explored, among which the inclusion in inorganic mesoporous matrixes, shown to be efficient and applied in the present work to menthol. An amorphous drug adsorbed on the surface of a porous silica matrix is stabilized via spatial confinement in small pores that limit drug nucleation and crystal growth when pore size lies below a critical dimension. Drug crystallization tendency may also be decreased by the development of drug−carrier interactions, which depend on the chemical surface of the matrix.13 In this context, ordered mesoporous silica materials are the object of increased interest due to their emerging applications as hosts in drug delivery (see ref 14 and references therein). MCM-41 and SBA-15 are among the most investigated materials of this type,15−18 having the same chemical composition, 100% silica, with uniform honeycomb structure formed by parallel cylindrical pores organized in a hexagonal array, with a narrow pore diameter distribution offering a large area for drug adsorption. These materials were used in the present work as menthol carriers, as a strategy to design a drug delivery system. For the pharmaceutical drugs ibuprofen19,20 and naproxen21 loaded in these mesoporous silica matrixes, dielectric relaxation spectroscopy (DRS) unraveled two molecular populations with different dynamical behavior: one with hindered mobility due to guest adsorption at the pore wall, and another one with accelerated mobility due to reorientational motions of guest molecules in the pore core. Furthermore, the knowledge of the relaxation times of the process that governs the dynamical glass transition (α-process), provided by DRS, is relevant to pharmaceutical science since it can be correlated to both crystallization onset and kinetics, allowing to predict drug stability.11,22,23 Another approach for the improvement of drug performance is the formation of a deep eutectic solvent (DES)4,24 by combination with another component. According to Abbot et al.,25 DES is a mixture of two crystalline solids that when mixed at specific molar ratios present a much lower melting point than the individual components. The recent inclusion of a drug as at least one of the eutectic mixture constituents has led to the concept of a therapeutic deep eutectic solvent (THEDES).26 THEDES represents a step further toward pharmaceutical and biomedical applications, due to their ability to increase drug solubility and to improve permeation and absorption of poorly water-soluble drugs.10,26−28 This permeation enhancement was observed for THEDES comprising ibuprofen and a terpene, such as menthol29−31 or camphor.31 In these previous studies, the target drug was ibuprofen, and menthol was used to enhance drug performance. However, these studies also revealed that the thermal behavior of menthol was influenced by the presence of the other drug, and in some cases to such an extent as to suppress menthol crystallization.

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EXPERIMENTAL SECTION Materials. (±)-Menthol ((±)-2-isopropyl-5-methylcyclohexanol; C10H20O), designated hereafter as menthol, was purchased from Sigma-Aldrich (catalogue number 63670, CAS number 89-78-1, >98.0% assay, molar mass of 156.27 g mol−1). Menthol possesses asymmetric carbon atoms and thus it can exist as two enantiomers, namely, levorotatory, L(−)-, and dextrorotatory, D(+)-menthol (Scheme 1). The menthol used Scheme 1. Chemical Structures of D(+)-Menthol and L(−)Menthol (Retrieved from Ref 32)

in the present work, DL(±)-menthol, was previously demonstrated to be a racemate.33 Its almost null specific optical rotation (−0.01°) was confirmed by preparing a 10% (w/v) solution of menthol in ethanol and measuring the optical rotation [α]D20 for (−)-menthol and (+)-menthol in a Bellingham Stanley (ADP 410) polarimeter, which led to the values −50° and +48°, respectively.34 (±)-Flurbiprofen ((±)-2-fluoro-α-methyl-4-biphenylacetic acid; C15H13FO2) was purchased from Sigma-Aldrich (catalogue number F8514, CAS number 5104-49-4, molar mass of 244.26 g mol−1) (Scheme 2). Scheme 2. Chemical Structure of Flurbiprofen (Retrieved from Ref 35)

(−)-Menthol (2-isopropy-5-methylcyclohexanol-1,2,6,6-d4; C10H16D4O), designated hereafter as isotopically labeled menthol, was purchased from Cambridge Isotope Laboratories (catalogue number NC0309156, ≥98% assay, molar mass of 160.29 g mol−1). 3165

DOI: 10.1021/acs.molpharmaceut.7b00386 Mol. Pharmaceutics 2017, 14, 3164−3177

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Molecular Pharmaceutics Menthol:Flurbiprofen THEDESs. Menthol was combined with flurbiprofen according to a previous report.26 All THEDESs were prepared by mixing the two components at the desired molar ratio and heating at 40 °C under constant stirring for 24 h. To ensure the correct molar ratio after the first DSC run where a maximum temperature of 135 °C was used, the mixture was weighed again to account for possible loss of menthol due to evaporation. The THEDESs studied are listed in Table 1.

glass cell under vacuum. The solvent was allowed to evaporate for 3 days under gentle stirring at 40 °C, after which a dry powder was obtained. Hereafter, the composites obtained will be designated according to the matrix as mentholMCM_3.2 nm and mentholSBA_5.9 nm. Techniques. Thermogravimetry Analysis (TGA). Samples of ∼3 mg (measured accurately) were placed in an open platinum sample pan, and the thermogravimetric measurements were carried out from 22.5 to 550 °C with a TGA Q500 apparatus from TA Instruments Inc., at a heating rate of 5 °C min−1 under highly neat nitrogen atmosphere with a sample purge flow rate of 60 mL min−1. The temperature reading was calibrated using the Curie points of nickel standard, while the mass reading was calibrated using balance tare weights provided by TA. TGA analysis was used to evaluate the percentage of loading of menthol in each silica matrix. The obtained weight percentage thermograms are shown in Figure 1a and compared with neat menthol.

Table 1. Summary of the Molar Ratios Used in THEDESs Preparation and Obtained after Correction for Menthol Mass Loss (Please See Text); x = mole fraction composition, given as xmenthol:xflurbiprofen

molar ratio, prepared

molar ratio, corrected

6.1:1 4.7:1 2.8:1 2.5:1 2.2:1

6.6:1 5.5:1 3.3:1 2.5:1 4.4:1

6.1:1 4.7:1 2.8:1 2.5:1 2.2:1

MCM-41 and SBA-15 Matrixes. The synthesis of MCM-41 and SBA-15 matrixes is described in ref 11 and was carried out accordingly.36,37 Briefly, for MCM-41, aerosil was used as a silica source and dodecyl trimethylammonium bromide as a template, while for SBA-15, tetraethyl orthosilicate was used as a silica source and the block copolymer poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide) was used as a template, under acidic conditions. Calcination at a heating rate of 1 °C min−1 to 500−550 °C was used to completely remove the organic template, followed by 6 h under dry air. Transmission electron microscopy (TEM) analysis confirmed the uniform honeycomb structure formed by parallel cylindrical pores organized in a hexagonal array in MCM-4138 and the two-dimensional hexagonally ordered cylindrical pores in SBA15.39 Nitrogen absorption analysis was used to obtain the textural features of MCM-41 and SBA-15 matrixes (see Table 2). The Table 2. Textural Properties of the Synthesized Mesoporous Silica Matrixes material

surface area (m2 g−1)

total pore volume (cm3 g−1)

pore diameter (nm)

MCM-41 SBA-15

998.0 719.3

0.883 0.981

3.2 5.9

specific surface area was determined from the linear portion of the Brunauer−Emmett−Teller (BET) plots, the total pore volume by the density functional theory (DFT) method, and the pore size by the Barrett−Joyner−Halenda (BJH) desorption method.40 Drug Loading. Menthol was dissolved in chloroform for incorporation into the two silica matrixes, performed under vacuum. To eliminate water and other impurities, which could be contained in the pores of silica, 100.2 mg of MCM-41 silica or 100.0 mg of SBA-15 silica, were placed in a glass cell under vacuum (10−4 bar) and heated up to 150 °C by immersion of the cell in a paraffin bath, for 8 h. After this period of time, the cells were allowed to cool down to room temperature and were kept under vacuum until use. Solutions of 71.6 mg (for MCM41 silica) or 72.8 mg (for SBA-15 silica) of menthol dissolved in 1.5 mL of chloroform were prepared and transferred to the

Figure 1. (a) Thermogravimetric curves obtained on heating at 5 °C min−1 for neat menthol and menthol-loaded matrixes. (b) Thermograms in the derivative plot.

Most of the menthol in the composites decompose at a higher temperature than neat menthol, which starts to decompose close to 100 °C. While the onset of the weight loss curves is slightly shifted to higher temperatures in the composites, more pronounced when the drug is loaded into the smaller pore size matrix, for both composites the endset occurs around 50 °C above the one for neat menthol. This indicates 3166


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Molecular Pharmaceutics that the inclusion in the inorganic matrixes improves the thermal stability of the guest, as reported for other drugs in homologous matrixes.17,21 A bimodal loss weight profile is found for the composites confirmed by the derivative representation (Figure 1b), contrary to neat menthol that decomposes in a single weight loss step. From the loss weight, menthol loading (% w/w) was determined according to eq 1a (see Table 3), taking into Table 3. Loading (eq 1a) and Filling (eq 1b) Percentages in Menthol/Silica Composites mentholMCM_3.2 nm mentholSBA_5.9 nm

loading % (w/w)

filling % (v/v)

64 73

81 84

account the drug loading, pore volume (see Table 2), and menthol density (0.89 g cm−3 at 25 °C),41 and the filling % (v/ v) was estimated according to eq 1b loading % =

mmenthol (g) × 100 msilica (g)

Figure 2. X-ray diffraction patterns for neat menthol, unloaded SBA matrix, mentholMCM_3.2 nm, and mentholSBA_5.9 nm, obtained at room temperature. Bragg peaks are only detected for neat, crystalline menthol, while the neat matrix and composites exhibit the halo characteristic of an amorphous form. The XRD pattern of unloaded SBA matrix was vertically displaced for better visualization.

(1a)

mmenthol (g)

filling % =

ρmenthol (g cm−3)

pore volume(cm 3g −1)msilica (g)

cm−1, which corresponds to the asymmetric stretching vibration, νas(Si−O−Si), of the siliceous framework. The symmetric stretch, νs(Si−O−Si), of the siliceous framework was observed at 800 cm−1 (see Figure 3a).16 These bands, together with the broad band at about 3400 cm−1 (stretching vibration of the physisorbed water) were observed in the spectra of all samples except for neat menthol (see Figure 3b). Instead, since menthol is a terpene alcohol, it displays a strong and broad peak at 327043/328144 cm−1, which is indicative of the −OH stretching vibration. Absorption due to C−H stretching was observed43 between 2800 and 2900 cm−1. Significant peaks were found at 2855, 2924,45 and 295344 cm−1, due to the stretching vibration of the methyl group (CH3),44,45 at 1025 and 1045 cm−1, attributed to the C−O bond,45 and 136745/136844 cm−1, corresponding to the isopropyl group stretching vibration. At lower frequencies (1375 to 1470 cm−1), menthol displayed carbon−hydrogen bending typical of methyl groups present in menthol.43 The appearance of peaks characteristic of both menthol and siliceous framework in the composites spectra, albeit less structured, indicate successful loading of menthol. Differential Scanning Calorimetry (DSC). The calorimetric experiments were performed using a DSC Q2000 from TA Instruments Inc. (Tzero DSC technology) operating in the Heat Flow T4P option. Measurements were carried out under anhydrous high purity nitrogen at a flow rate of 50 mL min−1. DSC Tzero calibration was carried out in the temperature range from −90 to 200 °C. It required two experiments: the first run with the empty cell (baseline), and the second run with equal weight sapphire disks on the sample and reference platforms (without pans). This procedure allows for cell resistance and capacitance calibration, which compensates for subtle differences in thermal resistance and capacitance between the reference and sample platforms in the DSC sensor. Enthalpy (cell constant) and temperature calibration were based on the melting peak of indium standard (Tm = 156.60 °C) supplied by TA Instruments (Lot #E10W029). Samples, with mass ∼3−5 mg, were encapsulated in Tzero (aluminum) hermetic pans with a Tzero hermetic lid with a pinhole to allow solvent and water evaporation.

× 100 (1b)

A high inclusion level was achieved for both composites. Moreover both loading and filling percentages are quite similar for the two composites, allowing a direct comparison of the drug release results. X-ray Diffraction (XRD). Powder X-ray diffraction analysis was carried out with a PANalytical X’Pert pro MPD diffractometer equipped with a Cu X-ray tube (selected wavelength λCuKα = 1.54056 Å) and the X’celerator detector. The samples were enclosed in a Lindemann glass capillary (diameter 0.7 mm), which was rotated during the experiments. The measurements were performed in transmission mode with incident beam parabolic mirror. To access the physical state of loaded menthol, the composites were analyzed through X-ray diffraction, whose patterns at room temperature are presented in Figure 2, which includes the powder X-ray diffraction patterns of neat menthol and unloaded SBA matrix. While distinct and sharp Bragg peaks are detected for neat menthol, arising from its crystalline structure (triclinic space group P1),33 for the neat matrix and composites only the halo characteristic of an amorphous form is observed. This acts as an evidence of the drug being fully amorphous in mentholMCM_3.2 nm and mentholSBA_5.9 nm. Bragg peaks become observable at small angles, 2° < θ < 10° due to the long-range order of the two-dimensional hexagonal symmetry of the mesoporous matrixes (not shown; for details see ref 42). Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Fourier transform infrared (FTIR) spectra over the range 650 to 4000 cm−1 were collected at room temperature using a Cary 630 FTIR spectrometer equipped with diamond attenuated total reflectance (ATR) from Agilent Technologies, with a thermoelectrically cooled dTGS detector and KBr standard beam splitter. All the spectra were recorded via ATR method with a resolution of 1 cm−1 and 16 scans. The FTIR spectrum of SBA_5.9 nm is characterized by the presence of a strong and broad band observed at about 1000 3167


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dispersed in 100 mL of release media in a glass flask placed in a horizontal shaker at 100 rpm, at a constant temperature of 37 °C. During the time course of each experiment, 1 mL sample was withdrawn at defined intervals, and the flask volume was replenished with the same volume of fresh medium. The samples were filtered with GHP Acrodisc Minispike (Φ = 0.45 μm, 13 mm) syringe filters supplied by Pall Life Sciences. The aqueous filtrate (0.5 mL) was mixed with 0.5 mL of aqueous solution of isotopically labeled menthol acting as an internal standard (IS). This solution was made up to 10 mL with deionized water and analyzed by headspace solid phase microextraction coupled with gas chromatography/mass spectrometry (HS-SPME-GC/MS). Quantification of menthol was achieved by the internal standard method. Calibration curves were obtained for a concentration range of 0.001−0.495 mg L−1 of menthol using isotopically labeled menthol as internal standard in a concentration of 0.439 mg L−1. The three calibration curves were linear with R2 = 0.9997; 0.9979; 0.9955. The drug release experiments were performed in multiple replicates. Headspace Solid Phase Microextraction (HS-SPME). The headspace solid phase microextraction (HS-SPME) was performed using a carboxen/divinylbenzene/polydimethylsiloxane fiber (CAR/DVB/PDMS, 1 cm, 50/30 μm film thickness (df)) supplied by Supelco, Bellefonte, PA, USA. Prior to use, the fiber was conditioned following the manufacturer’s recommendations. Fiber blanks were run periodically to ensure the absence of contaminants and/or carryover. The samples, with 10 mL volume each, were introduced in a 22 mL vial and sealed with a Teflon-lined rubber septum/magnetic screw cap. The vial was equilibrated for 10 min at 60 °C and then extracted for 20 min at the same temperature. Thermal desorption of the analytes was carried out by exposing the fiber in the GC injection port at 260 °C for 3 min in splitless mode. Gas Chromatography/Mass Spectrometry-Selected Ion Monitoring (GC/MS-SIM) Analysis. The analyses were performed on a GC/MS system consisting of a Bruker GC 456 with a Bruker Scion TQ mass selective detector. An automatic sampler injector was used: CTC Analysis autosampler CombiPAL. Data were acquired with MSWS 8.2 Bruker and analyzed with Bruker MS Data Review 8.0. Chromatographic separation was achieved on a ZB-WAX PLUS capillary column (30 m × 0.25 mm i.d., 0.25 μm df) connected to a ZB5 MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film df), both supplied by Phenomenex, Torrance, CA, USA. In order to perform flow control a third deactivated capillary column was used (30 m × 0.25 mm i.d.), supplied by Bruker, Billerica, MA, USA. All three columns were connected by means of a Valco VICI−Stainless steel tee 1/32″ (Tubing OD), 0.25 (Bore) Dean’s switch, VICI, AG, International, Schenkon, Switzerland. The oven temperature program was plateau at 80 °C for 1 min, followed by a 4 °C min−1 ramp to 155 °C, then a 20 °C min−1 ramp to 240 °C, and hold for 5 min. Helium was used as carrier gas, at a constant pressure of 35.0 Psi at the electronic flow control 21 (EFC 21) and 23.0 Psi at the EFC 24. The MS transfer line and source temperatures were set at, respectively, 240 and 220 °C. Spectra were matched with NIST MS Search Program Version 2.0g. To determine the retention times and characteristic mass fragments, electron ionization (EI) at 70 eV mass spectra of the analytes were recorded at full scan, from 40 to 250 u. When single ion monitoring (SIM) mode was used for quantitation, m/z 138 for menthol and 142 for menthol-d4

Figure 3. ATR-FTIR spectra for neat menthol, empty SBA matrix, and composites: (a) from 400 to 2500 cm−1 and (b) from 2500 to 4000 cm−1. The superposition of menthol and silica absorption peaks in the composite spectra is an indication of successful loading. All the curves were vertically displaced for better visualization.

The sample of neat flurbiprofen was cooled down from 40 to −80 °C followed by two heating cycles between −80 and 135 °C, at 10 °C min−1. The sample of neat menthol was submitted to a heating scan from 18 to 80 °C followed by two heating cycles between −90 and 80 °C, at 10 °C min−1. The menthol:flurbiprofen samples were submitted to two cooling and heating runs between −80 and 80 °C at 10 °C min−1, after a first cycle between −80 and 135 °C. The samples of the mentholMCM_3.2 nm and mentholSBA_5.9 nm composites were cooled down to −90 °C and heated up to 80 °C in the first scan and kept at this temperature during 20 min for water removal. Then the samples were submitted to annealing by being maintained for 90 min at −55 °C and were then cooled down to −90 °C, followed by heating up to 90 °C at a rate of 30 °C min−1. Menthol Release Experiments. The experiments of in vitro menthol release from silica matrixes were conducted in phosphate buffer at pH 6.8 (simulated intestinal fluid). One milligram of neat menthol (in dissolution studies) or the amount of a silica matrix containing 1 mg of menthol was 3168



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were used as quantifier ions and 123 for menthol and 127 for menthol-d4 as qualifier ions. Dielectric Relaxation Spectroscopy (DRS). Dielectric measurements were carried out using the ALPHA-N impedance analyzer from Novocontrol Technologies GmbH, covering a frequency range from 10−1 Hz to 1 MHz. A small amount of powder composite was slightly compressed between two gold plated electrodes (10 mm of diameter) of a parallel plate capacitor, BDS 1200, with two 50 μm-thick silica spacers. The sample cell was mounted on a BDS 1100 cryostat and exposed to a heated gas stream evaporated from a liquid nitrogen Dewar. The temperature control was ensured by Quatro Cryosystem and performed within ±0.5 °C. Novocontrol Technologies GmbH supplied all these modules. To remove water, the samples were annealed for 10 min at 80 °C in the sample cell, after which they were cooled down to −90 °C. The isothermal dielectric spectra were collected between −90 and 80 °C at different increasing temperature steps of 2 °C in the range −80 to 80 °C, and of 5 °C in the remaining temperature range. No measurements or water removal were carried out at higher temperatures to avoid menthol sublimation. Dielectric Data Analysis. To analyze the isothermal dielectric data, the model function introduced by Havriliak− Negami (HN)46,47 was fitted to both imaginary (ε″) and real components (ε′) of complex permittivity (ε*(ω) = ε′(ω) − iε′(ω)) when conductivity and electrode polarization did not affect the spectra significantly. Because multiple peaks are observed in the available frequency window, a sum of HN functions was employed:

∑

ε*(ω) = ϵ∞ +

j

Figure 4. Thermograms of neat menthol scanned at a heating rate of 10 °C min−1. Each run is depicted in the same color and line type as in the temperature treatment scheme (included in the inset).

In the first heating scan only melting is observed by the detection of a sharp endothermic peak centered at 39.2 °C (respective melting enthalpy, ΔHm = 96 J g−1; dashed red line in Figure 4). In the subsequent cooling run from the melt, a complex exothermic peak is clearly seen (solid blue line) due to menthol crystallization. In the following heating scan two endothermic peaks are detected at 28.1 and 34.3 °C (solid green line). This calorimetric behavior is in close agreement with the one reported for both L-menthol and DL-menthol (racemate) where two polymorphs, designated as β and α, were identified. In the case of DL-menthol, the reported33 melting temperatures, respectively, 27.3 and 33.8 °C, are in close agreement with those detected here. The typical discontinuity in the heat flow acting as the signature of the glass transition cannot be seen, indicating that below the melting temperature menthol is fully crystalline. The subsequent runs reproduced this behavior indicating that (a) menthol exhibits polymorphism and that (b) it is not able to circumvent nonisothermal crystallization under the applied thermal treatment. Thermal Behavior of Menthol:Flurbiprofen THEDESs. The main goal of using THEDESs in this work was then to take advantage of the thermal behavior of these compounds as an alternative to avoid menthol crystallization and allow the estimation of its glass transition temperature, Tg. First, the phase transformations of flurbiprofen were analyzed by DSC (see Figure 5). In the first heating run (red line in Figure 5), a sharp endothermic peak due to flurbiprofen melting is detected, centered at 114.9 °C. Contrary to menthol, no thermal events are detected for flurbiprofen in the subsequent cooling run, and consequently, no melt-crystallization occurs, which indicates that the material became full amorphous (blue line in the inset;

(2)

where j is the index over which the relaxation processes are summed, Δε is the dielectric strength, τHN is the characteristic HN relaxation time, and αHN and βHN are fractional parameters (0 < αHN < 1 and 0 < αHNβHN < 1) describing, respectively, the symmetric and asymmetric broadening of the complex dielectric function.48 From the τHN, αHN, and βHN parameters estimated from the fitting, a model-independent relaxation time, τmax = 1/(2πf max), was determined according to48

τmax

⎡ ⎢ sin = τHN⎢ ⎢ sin ⎣

( (

αHNβHNπ 2 + 2βHN αHNπ 2 + 2βHN

) )

⎤1/ αHN ⎥ ⎥ ⎥ ⎦

(3)

The nonlinear temperature dependence of the relaxation times can be described by the well-known Vogel−Fulcher− Tammann−Hesse (VFTH) equation49−51 as ⎛ B ⎞ τ(T ) = τ∞ exp⎜ ⎟ ⎝ T − T0 ⎠

RESULTS AND DISCUSSION

Thermal Behavior of Neat Menthol. Differential scanning calorimetry was used to study the phase transformations of neat menthol. Figure 4 shows the respective DSC thermograms (see Experimental Section for details and inset in the figure with the temperature treatment scheme).

Δεj [1 + (iωτHNj)αHNj ]βHNj

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(4)

where τ∞ is the value of the relaxation time in the high temperature limit, B is an empirical parameter characteristic of the material, accounting for the deviation from linearity (roughly, the lower the B, the more curved the 1/T plot), and T0 is the Vogel temperature, which can be related to the glass transition temperature of an ideal glass, i.e., a glass obtained with an infinitely low cooling rate.52 3169


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135 °C. In the recalculation, it was assumed that the mass difference only affects the menthol mass since the onset of its sublimation/degradation takes place near 100 °C, as observed by TGA (see Figure 1), while flurbiprofen has higher thermal stability, exhibiting 99.7% of degradation between 130 and 260 °C.53 The end temperature of 135 °C in the first cycle was chosen to confirm if any flurbiprofen-rich fraction exists in the prepared THEDESs, as this neat constituent melts at ∼115 °C after undergoing a glass transition slightly below 0 °C. In fact, both a slight melting peak close to the melting temperature of neat flurbiprofen and a small step due to a glass transition slightly below 0 °C are observed in the first run of some THEDESs, as illustrated for the 2.8:1 mixture (see solid line in the inset in Figure 6). Both thermal events disappear in the subsequent heating run (dashed line in the inset), evidencing that homogenization is only achieved after heating above the highest melting temperature of the neat constituents. Only a distinct step in the heat flow remains below −40 °C due to the glass transition. The following runs were taken only up to 100 °C with no further mass loss and keeping invariant the temperature location of the glass transition. The values of the glass transition temperatures taken from the onset and the midpoint of the heat flux step detected in the last heating run in all thermograms are presented in Table 4.

Figure 5. Thermograms of neat flurbiprofen scanned at a heating rate of 10 °C min−1. Each run is depicted in the same color as the respective trace in the temperature treatment scheme (included in the inset).

not shown in the main figure). The respective glass transition clearly emerges in the second heating run slightly below 0 °C, followed by cold crystallization and melting (green line). The melting endotherm superimposes with the one observed in the first cycle. This behavior is reproduced in the following cooling/heating run (not shown) indicating that flurbiprofen easily avoids crystallization upon cooling from the melt. Figure 6 exhibits the DSC thermograms (last heating run) for the menthol:flurbiprofen THEDESs in five different molar

Table 4. Tg onset and Tg midpoint Determined from the DSC Last Heating Run, Obtained at a Heating Rate of 10 °C min−1, for Neat Flurbiprofen and Menthol:Flurbiprofen THEDESs, Expressed as Both Final Menthol:Flurbiprofen Molar Ratio and Flurbiprofen Mole Fraction Tg onset

Tg midpoint

xmenthol:xflurbiprofen

xflurbiprofen

°C

K

°C

K

0 6.1:1 4.7:1 2.8:1 2.5:1 2.2:1

1 0.311 0.288 0.262 0.176 0.141

−3.73 −47.64 −45.59 −43.45 −42.46 −40.80

269.44 225.51 227.56 229.70 230.69 232.35

−3.06 −45.61 −44.18 −41.91 −40.88 −39.82

270.19 227.54 228.97 231.24 232.27 233.33

Additionally, three peaks representing first order transitions, namely, a broad exothermic peak due to cold-crystallization and two endothermic peaks corresponding to melting, can be observed in the thermograms for the 4.7:1 and 6.1:1: mixtures. Therefore, in spite of all combinations being fully amorphous upon cooling, the 4.7:1 and 6.1:1 ones are able to recrystallize when heated above the glass transition (cold crystallization). Melting in these menthol-rich THEDESs is observed slightly below 30 °C in the form of a two peak profile similarly to that for neat menthol. This can be interpreted as menthol governing the crystallization, regardless of melting occurring in a temperature range lower than any of those of the single components. The observed decrease in melting temperature is the expected behavior of a eutectic mixture according to the definition of Abbot et al.25 (see Introduction). When working above the liquidus line of the mixture, only one liquid phase exists. In the case of some DES or THEDES at specific molar ratios, melting can be completely suppressed, and a metastable amorphous phase below the equilibrium liquidus line is obtained as observed here for the remaining compositions. Interestingly, the glass transition in the last heating run, located slightly below −40 °C, shifts continuously to lower

Figure 6. Thermograms of menthol:flurbiprofen THEDESs obtained at a heating rate of 10 °C min−1. Thermograms were vertically displaced to allow a better visualization. The 1st heating run of the 2.8:1 THEDES is presented in the inset (solid line), evidencing the flurbiprofen-like glass transition and melting (see arrows) that disappear in the 2nd heating run (dashed line).

ratios of 2.2:1; 2.5:1; 2.8:1; 4.7:1; and 6.1:1, taken at a rate of 10 °C min−1. After the complete thermal treatment, all samples were immediately reweighed. Therefore, the molar ratios here provided were obtained after correction for the mass loss. This only occurred in the first heating run, which was carried up to 3170


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regression extrapolation to xflurbiprofen = 0.0, as −54.3 °C. This is a reasonable temperature value since from the variation of the Tgs determined for the different THEDESs, a Tg value of neat menthol below −40 °C was expected. Menthol Loaded in Mesoporous Silica. In this section, the approach employed to obtain neat-like amorphous menthol by loading into mesoporous MCM-41 (3.2 nm) and SBA-15 (5.9 nm) silica is explored in detail and studied with several techniques. Differential Scanning Calorimetry (DSC). The composites prepared by loading menthol into the two silica matrixes were calorimetrically analyzed. Figure 8 shows the thermograms collected in the first heating run (red solid line) for menthol loaded in (a) MCM_3.2 nm and (b) SBA_5.9 nm, where no melting peak is observed. The only event detected is the beginning of an endothermal peak due to water evaporation. In spite of the drying process to which the matrixes were submitted, silica readily adsorbs water during handling. Full water removal by heating silica at higher temperatures was not carried out since menthol readily decomposes (see thermogravimetric analysis in Figure 1). Instead, the samples were kept for 20 min at 80 °C (see Experimental Section). In the subsequent cooling run down to −90 °C (blue solid line), no sign of recrystallization appears, at least under nonisothermal conditions, and thus menthol must be in the supercooled/ amorphous state. To detect the glass transition, samples of each composite were submitted to annealing during 90 min at −55 °C, after which they were cooled down to −90 °C. Next, a heating scan up to 90 °C at a rate of 30 °C min−1 was performed. In the resulting thermogram the discontinuity in the heat flux clearly emerges, with the onset located at ∼−47 °C, unraveling the glass transition in both composites (see arrows in Figure 8a,b and respective scale up in Figure 8c). This acts as a further confirmation that menthol is amorphous/supercooled inside the pores. In fact, in the XRD diffraction patterns of both composites, shown in the Experimental Section, only the halo characteristic of the amorphous matrix and amorphous guest was detected. Dielectric Relaxation Spectroscopy (DRS). The mobility associated mainly with the dynamic glass transition of amorphous menthol regions inside pores was analyzed by DRS. Figure 9 shows the loss spectra for (a) mentholMCM_3.2 nm and (b) mentholSBA_5.9 nm collected upon heating at some representative temperatures. It must be noted that the dielectric loss of the silica matrix host after water removal is negligibly small,20 compared to that observed for the loaded matrixes. Therefore, the detected dielectric response is due to the guest menthol molecules. Although a relatively weak dielectric response is measured for the composites, it allows the resolution in distinct peaks. These become also evident in the isochronal plot represented at a frequency of 100 Hz in Figure 10. Furthermore, the ε″(T) trace shows no abrupt discontinuity in the region of the melting (between 20 and 40 °C) indicating that no crystallization occurred during DRS data acquisition. The same happens for the ε′(T) trace (not shown), and so, as concluded from the DSC analysis of the composites, menthol remains amorphous/ supercooled inside the pores. As mentioned in the Experimental Section, the model function of Havriliak−Negami (HN function, eq 2) was used to analyze the data and to resolve the relaxation processes. In both composites a bimodal behavior is recognizable, more accentuated for mentholSBA_5.9 nm, and thus, the spectra were

temperatures with the increase in menthol content (see Table 4). Since no glass transition could be observed in the DSC thermogram for neat menthol, the values of glass transition temperatures obtained for the menthol:flurbiprofen THEDESs were analyzed by the Fox equation (eq 5a). This analytical equation predicts intermediate values of the glass transition for an ideal binary mixture as a function of the mole fraction of the neat constituents.54 Tg,mix =

1 Tg,mix

=

Tg1Tg2 x1Tg2 + (1 − x1)Tg1

x1 1 − x1 + Tg1 Tg2

(5a)

(5b)

For a binary system, Tg,mix refers to the mixture, while Tgi refers to neat component i, xi being its mole fraction. The validity of the Fox equation in the description of the behavior of the binary menthol:flurbiprofen THEDESs was tested by using the glass transition temperatures extracted from the onset of the heat flux discontinuity. The Fox equation describes relatively well the composition dependence of the THEDESs glass transition temperature, as seen by the plots of eq 5a in Figure 7 and eq 5b in the inset.

Figure 7. Plot of the Fox equation (eq 5a) for menthol:flurbiprofen THEDESs. In the inset, the linear plot 1/Tg, ons_mix vs flurbiprofen mole fraction (eq 5b) is presented (1/Tg,ons_mix = −8.51 × 10−4 xflurbiprofen + 4.57 × 10−3, r2 = 0.997). In both figures, the star indicates the glass transition temperature predicted by DRS measurements at τ = 100 s for neat menthol (see text).

Previous NMR studies55 from members of the research group on THEDESs comprising menthol and an homologous profen drug carrying a carboxylic acid, ibuprofen, showed that the hydroxyl groups of both menthol and the acid were in fast exchange and that the intermolecular bonding was similar in all cases and independent of the carboxylic acid. This suggested that the intermolecular bond energies menthol−menthol, carboxylic acid−carboxylic acid, and menthol−carboxylic acid are similar. This fact and the similarity between ibuprofen and flurbiprofen lead us to expect no significant deviations from the Fox equation for menthol:flurbiprofen THEDESs as here observed. This can also be in the origin of the crystallization avoidance under the applied thermal treatment. The application of the Fox equation allows estimation of the Tg value of neat menthol by taking the reciprocal of the linear 3171


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Figure 9. Isothermal loss spectra for the two composites: (a) mentholMCM_3.2 nm and (b) mentholSBA_5.9 nm, at some representative temperatures. The dashed lines are the individual HN functions considered for simulation of the spectrum at −40 °C for mentholMCM_3.2 nm and at −20 °C for mentholSBA_5.9 nm. For the latter the conductivity contribution was also considered. The solid lines represent the overall fit for each isotherm. The small vertical bars indicate the frequency location of the peak of the α-process at −40 °C for both composites.

Figure 10. Isochronal plot at 100 Hz for the two composites enhancing the broadness of the peak of mentholMCM_3.2 nm and the bimodal nature of the dielectric response of mentholSBA_5.9 nm. The location of the underlying α- and S-processes is indicated.

Figure 8. Thermograms of menthol loaded into (a) MCM_3.2 nm and (b) SBA_5.9 nm matrixes, scanned at 10 °C min−1. The thermograms represented by a dashed red line were obtained at a heating rate of 30 °C min−1 after annealing for 90 min at −55 °C, revealing the glass transition (see arrow in each panel). This transition, which is scaled-up in panel (c), has an onset at −47.5 °C for both composites (Tg,mid_MCM_3.2 nm = −40.0 °C, Tg,end_MCM_3.2 nm = −34.8 °C, Tg,mid_SBA_5.9 nm = −43.4 °C, Tg,end_SBA_5.9 nm = −39.5 °C).

well simulated using two HN processes (shape parameters are included in Table 5) and conductivity. It is worth noting that in the TGA analysis the mass loss occurred also according to a 3172


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Table 5. Shape Parameters Obtained by Fitting the HN Equation (eq 2) to the Isothermal Spectra for the Main Processes Detected in the Two Composites and VFTH Fitting Equation Parameters (eq 4) process

αHN

βHN

τ∞ (s)

B (K)

T0 (K)

Tg, diel τ = 100 s (K)

αSBA_5.9 nm αMCM_3.2 nm SSBA_5.9 nm SMCM_3.2 nm

0.47 → 0.67 0.59 0.33 ± 0.03 0.36 → 0.58

0.61 0.50 0.8 0.48 → 0.34

1.2 × 10−18

2868

155.0

217.6 (−55.6 °C)

4.5 × 10−17

3612

137.2

222.7 (−50.4 °C)

process is associated with the glass transition of menthol bulklike molecules and hence its designation as a α-process. Regarding the process detected at lower frequencies for menthol in the 5.9 nm-pore matrix, it is clear that the estimated relaxation times derived from either isothermal or isochronal fits fall in one chart. The higher relaxation time for this relaxation process indicates that the guest molecules relax with a slower relaxation rate. This is due to adsorption at the pore wall, hindering the dipolar reorientational motion. This process is usually named the surface (S) process and is also found in the pharmaceutical drugs ibuprofen19,20 and naproxen21 incorporated in similar silica matrixes, for which the more mobile bulklike α-process is observed as well. It is worth noting that the S-process has a higher magnitude compared with the α-process in both composites (see dashed lines of the individual HN functions used to simulate the overall dielectric response in Figure 9). However, this behavior is much more accentuated in mentholMCM_3.2 nm, indicating that the dynamical behavior in the smaller pore size is highly dominated by the fraction of adsorbed molecular population: e.g., while at −30 °C the ratio between the dielectric strength (Δε) of the Sand α-processes in mentholMCM_3.2 nm is 0.144/0.013 (∼11), in mentholSBA_5.9 nm the ΔεS/Δεα is 0.086/0.047 (∼2). The isochronal plot illustrates well the ratio between both molecular populations (see Figure 10) where two distinct peaks are defined for mentholSBA_5.9 nm, while a broad one with a small shoulder in its low temperature side is observed for mentholMCM_3.2 nm. It is interesting to note that, at least at the lowest temperatures, the wide loss peaks of mentholMCM_3.2 nm can be simulated by a combination of the αand S-processes with the same location found for mentholSBA_5.9 nm. In Figure 9, it is evident the closeness of the maxima of the α-peaks for the two composites at −40 °C, as indicated by the small vertical bars. Due to a progressive depletion of the α-process with the temperature increase, as found for the cooperative process that drives the dynamical glass transition in conventional glass formers, the accuracy in determining the location of this process for menthol in the lower pore size matrix becomes poor at the highest temperatures. This is the reason why the VFTH analysis of the αprocess was carried out only for mentholSBA_5.9 nm. Concerning the S-process, the dominance of the population that relaxes with a slower relaxation rate seems to determine the calorimetric behavior. In fact, the extrapolated temperature dependence of the dielectric relaxation times of the S-process taking into account both composites and isothermal and isochronal data gives an estimation of the glass transition temperature (−50.4 °C) in closer agreement with the onset of the glass transition observed in DSC measurements (−47.5 °C) than the α-process (see arrow in Figure 11). Although the curvature exhibited by the temperature dependence of relaxation times of the S-process already indicates the cooperative nature of the underlying relaxation process, the agreement between the TgS,τ=100s and the calorimetric Tg acts as

two-step profile, indicative of two molecular populations inside pores. For both composites, in the lowest temperature range and high frequencies, a third relaxation process due to a secondary relaxation (aux-process in Figure 9) was used in the spectral simulation. However, it never showed up as a distinct process, and thus, no data is provided. From the HN fit (fit shape parameters in Table 5), the relaxation time of the different processes, τHN, was extracted and plotted in Figure 11 against the temperature reciprocal after conversion to τmax through eq 3 (circles).

Figure 11. Dependence of the logarithm of the relaxation times on the temperature reciprocal for the α- and S-processes detected in each composite (see legend inside figure). The solid lines are the respective VFTH fits (eq 4). For the S-process, both isothermal and isochronal data of mentholSBA_5.9 nm were considered together with the isothermal data for mentholMCM_3.2 nm. For the α-process, the VFTH fit is obtained with only the isothermal and isochronal data for mentholSBA_5.9 nm (the uncertainty affecting the relaxation times estimated for mentholMCM_3.2 nm is given by the magnitude of the error bars). The arrow indicates the temperature of the onset of the glass transition detected by DSC for both composites.

The relaxation times for the two main processes obey the VFTH law, as usually observed for the cooperative glassy dynamics. The extrapolation of the relaxation times to τ = 100 s gives an estimate of the glass transition temperature.56 The obtained dielectric Tg for the more mobile process in mentholSBA_5.9 nm is −55.6 °C, which fits quite well (see star in Figure 7) with the extrapolation of the linear fit describing the composition dependence of the THEDESs glass transition temperature. Since the Fox equation assumes an ideal behavior, i.e., that the menthol−menthol interactions are comparable to menthol− flurbiprofen and flurbiprofen−flurbiprofen interactions, it is reasonable to assume that the predicted glass transition temperature (−54.3 °C) for menthol is a good approximation of the Tg value of neat menthol. This confirms that the detected 3173


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melting temperature and enthalpy (Tm = 39.2 °C/312.25 K and ΔHm = 96 J g−1) are for menthol as received, whose respective thermal response was included in the thermogram of Figure 4. Figure 12 illustrates the temperature dependence of the nuclei diameter, evidencing that the 3.2 nm pore size is smaller than the critical size estimated for menthol nucleation. Thus, crystallization of menthol in mentholMCM_3.2 nm is inhibited due to small pore dimensions, in addition to slowed down dynamics toward the stabilization of the amorphous/supercooled state.13 However, when loaded into 5.9 nm pore matrixes, and although no crystallization of menthol is observed, the Gibbs−Thomson equation predicts that crystallization is inhibited only if the composite is maintained below ∼240 K (−33 °C). Drug Release Analysis. Aiming to evaluate how the drug is released from the matrix, delivery essays were conducted at pH 6.8 to simulate intestinal fluid and quantified by HS-SPMEGC/MS in SIM mode. It is worth noting that this represents unfavorable conditions since menthol at this pH is undissociated (pKa = 19.55),61 which does not favor its dissolution, although the assays were conducted below the reported value for the menthol solubility in water (0.456 g L−1 at 25 °C).62 The percentage of menthol released vs time at pH 6.8 is plotted in Figure 13 for the two composites, normalized for the

further confirmation that it is associated with the dynamic glass transition of the adsorbed menthol molecules. The calorimetric glass transition occurs over a quite broad heat flux step (see Figure 8c) due to the mixed behavior of different molecular populations. Therefore, DRS proved to be a suitable tool to deconvolute, mainly in the higher pore size matrix, the dynamical behavior of menthol molecules incorporated in the silica pores. The strong influence of the hindered population in the detected calorimetric glass transition and the abnormally wider heat flux step was also found for naproxen inside identical silica matrixes.21 The lower glass transition temperature estimated here for the bulk menthol α-process means that, even after the S-process becomes frozen, there is some mobility that survives and relaxes according to the α-relaxation. Therefore, the T0 value obtained for the α-process should be the one to take into account when predicting storage conditions. In fact, it is assumed that the relaxation times associated with the dynamic glass transition become so long at the Kauzmann temperature (and also at T0) that the molecular motions that govern instabilities can be considered negligible. This allows to establish a temperature limit for safe preservation below which the time scale for structural relaxation is of the order of years.57 To gain an additional insight of the behavior of menthol inside pores and its resistance to crystallization, which was mostly studied under nonisothermal conditions, it is possible to estimate a critical length scale for crystallization from the Gibbs−Thomson equation expressed in terms of the nuclei diameter (see ref 58 and references therein):

d=

4Tmσ ΔHmρc (Tm − T )

(6)

where Tm and ΔHm are, respectively, the melting temperature and enthalpy of the native compound, σ its surface tension, and ρc the solid density. The critical diameter estimated by eq 6 is plotted in Figure 12 for a surface tension of 0.0301 N m−1, an average value between the ones provided in refs 59 and 60. Since the menthol used to load silica matrixes was not previously submitted to any thermal treatment, the values of Figure 13. Release profiles of menthol from the composites in a simulated intestinal fluid (pH = 6.8). Lines are merely guiding lines.

% loading obtained by TGA. The dissolution of native menthol was evaluated under the same conditions, and the respective time dependence is included in the same figure for comparison. It is important to note that in the case of neat menthol the amount dissolved is rather low, only reaching a value slightly above 20% after 3 h. This is far below the reported solubility value,62 and thus, the detected amount of drug released is limited by the dissolution of the neat drug itself under these experimental conditions. Therefore, by comparing the amount released by the silica drug carriers with neat menthol dissolution, it seems reasonable to conclude that no guest is retained inside the matrixes pores. Moreover, a significant enhancement of the release of menthol from the composites at short times is observed, mainly for menthol loaded into the 5.9 nm silica matrix. Through DSC and DRS experiments, it was demonstrated that menthol is amorphous inside pores, with a molecular population that is partitioned between a fraction with slower relaxation rate and another one with bulk-like mobility. The fraction of menthol molecules with bulk-like mobility is

Figure 12. Temperature dependence of critical diameter of nuclei for menthol crystallization according to eq 6. Tm and ΔHm estimated from the melting of menthol as received. 3174


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from FCT/MEC (UID/QUI/50006/2013) and cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI01-0145-FEDER-007265). T.C. and M.T.V. acknowledge Fundaçaõ para a Ciência e a Tecnologia for the scholarships SFRH/BD/114653/2016 and SFRH/BPD/110151/2015, respectively. Nitrogen absorption analysis was obtained at Laboratório de Análises/Requimte of the Chemistry Department−Universidade Nova de Lisboa (http://www.dq.fct.unl. pt/en/analytical-services).

higher in mentholSBA_5.9 nm, the composite that exhibits faster release, reaching a plateau within the first 30 min. However, mentholMCM_3.2 nm needs more than 2 h to produce a comparable menthol concentration within the experimental error. This behavior reflects the hindrance in release imposed by the dominant population adsorbed at the inner pore walls. The faster release for mentholSBA_5.9 nm can be taken as an indication of the solubility improvement provided by a higher fraction of amorphous menthol with enhanced mobility, together with a high filling percentage (84% v/v). Therefore, the dynamical characterization provided by DRS allowed establishing a direct correlation between in situ guest mobility and the respective release profile.

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(1) Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability. Pharm. Res. 1995, 12, 413−420. (2) Kerns, E. H.; Di, L. Toxicity. In Drug-like Properties: Concepts, Structure Design and Methods from ADME to Toxicity Optimization, 1st ed.; Elsevier: London, 2008; pp 215−223. (3) Metcalfe, C.; Miao, X.-S.; Hua, W.; Letcher, R.; Servos, M. Pharmaceuticals in the Canadian Environmental. In Pharmaceuticals in the Environment. Sources, Fate, Effects and Risks, 2nd ed.; Kümmerer, K., Ed.; Springer: Heidelberg, 2004; pp 67−90. (4) Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N.; Pouton, C. W.; Porter, C. J. H. Strategies to address low drug solubility in discovery and development. Pharmacol. Rev. 2013, 65, 315−499. (5) Rades, T.; Gordon, K. C.; Graeser, K. Molecular Structure, Properties, and States of Matter. In Remington: Essentials of Pharmaceutics, 1st ed.; Felton, L., Eds.; Pharmaceutical Press: London, 2013; pp 177−206. (6) Craighead, D. H.; Alexander, L. M. Topical menthol increases cutaneous blood flow. Microvasc. Res. 2016, 107, 39−45. (7) Mirokuji, Y.; Abe, H.; Okamura, H.; Saito, K.; Sekiya, F.; Hayashi, S.; Maruyama, S.; Ono, A.; Nakajima, M.; Degawa, M.; Ozawa, S.; Shibutani, M.; Maitani, T. The JFFMA assessment of flavoring substances structurally related to menthol and uniquely used in Japan. Food Chem. Toxicol. 2014, 64, 314−321. (8) Damonte, S. P.; Selem, C.; Parente, M. E.; Ares, G.; Manzoni, A. V. Freshness evaluation of refreshing creams: Influence of two types of peppermint oil and emulsion formulation. J. Cosmet. Sci. 2011, 62, 525−533. (9) Gupta, P.; Kakumanu, V. K.; Bansal, A. K. Stability and Solubility of Celecoxib − PVP Amorphous Dispersions: A Molecular Perspective. Pharm. Res. 2004, 21, 1762−1769. (10) Serajuddin, A. T. M. Solid Dispersion of Poorly Water-Soluble Drugs: Early Promises, Subsequent Problems, and Recent Breakthroughs. J. Pharm. Sci. 1999, 88, 1058−1066. (11) Craig, D. Q. M.; Royall, P. G.; Kett, V. L.; Hopton, M. L. The relevance of the amorphous state of pharmaceutical dosage forms: glassy drugs and freeze dried systems. Int. J. Pharm. 1999, 179, 179− 207. (12) Lee, E. H. A practical guide to pharmaceutical polymorph screening & selection. Asian J. Pharm. Sci. 2014, 9, 163−175. (13) Knapik, J.; Wojnarowska, Z.; Grzybowska, K.; Jurkiewicz, K.; Stankiewicz, A.; Paluch, M. Stabilization of the Amorphous Ezetimibe Drug by Confining Its Dimension. Mol. Pharmaceutics 2016, 13, 1308−1316. (14) Moritz, M.; Geszke-Moritz, M. Mesoporous materials as multifunctional tools in biosciences: Principles and applications. Mater. Sci. Eng., C 2015, 49, 114−151. (15) Guo, Z.; Liu, X.-M.; Ma, L.; Li, J.; Zhang, H.; Gao, Y.-P.; Yuan, Y. Effects of particle morphology, pore size and surface coating of mesoporous silica on Naproxen dissolution rate enhancement. Colloids Surf., B 2013, 101, 228−235. (16) Halamová, D.; Badaničová, M.; Zeleňaḱ , V.; Gondová, T.; Vainio, U. Naproxen drug delivery using periodic mesoporous silica SBA-15. Appl. Surf. Sci. 2010, 256, 6489−6494.

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CONCLUSIONS The amorphization of menthol was successfully achieved through loading in two mesoporous silica matrixes as well as through formation of a therapeutic deep eutectic solvent (THEDES) with flurbiprofen. Menthol:flurbiprofen THEDESs in final molar ratios between 2.2:1 and 6.1:1 were studied by DSC and found to exhibit a glass transition. From the extrapolation of the Fox equation that describes the composition dependence of the glass transition temperature (Tg) in ideal binary systems, the Tg of neat menthol was estimated as −55.6 °C. While nonisothermal crystallization emerges in the 4.7:1 and 6.1:1 menthol:flurbiprofen THEDESs, it is completely suppressed when menthol is included in mesoporous silica hosts, the glass transition being the only event detected by calorimetry. The mobility of the molecular guest was probed by dielectric relaxation spectroscopy, which unraveled two molecular populations: a dominant one with hindered mobility due to adsorption at the inner pore walls, and another one with enhanced mobility, associated with molecules relaxing in the pore core. In the higher pore size (5.9 nm) matrix, the more mobile molecules give rise to a distinct relaxation process from which a glass transition temperature is estimated, in very good agreement with the one obtained by the THEDESs approach and thus due to the reorientational motions of amorphous bulk-like menthol. This molecular population seems to govern menthol release from the matrix, which reaches a maximum value within 30 min. However, a maximum delivery value is only achieved with the smaller pore size (3.2 nm) matrix after more than 2 h. This can be taken as an indication of solubility enhancement imparted by amorphous menthol lying in the center of the pores. However, the lower pore size inhibits crystallization since it is inferior to the critical size of menthol nuclei. Therefore, these composites seem to be suitable for use as drug delivery systems where the drug delivery profile can be adjusted by tuning the host pore dimensions.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Madalena Dionisio: 0000-0002-1487-0889 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Associate Laboratory for Green Chemistry LAQV, which is financed by national funds 3175


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