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Structural and Dynamical Properties of Indomethacin Molecules Embedded within the Mesopores of SBA-15: A Solid-State NMR View Tina Ukmar,† Tomaž Č endak,† Matjaž Mazaj,† Venčeslav Kaučič,†,‡ and Gregor Mali*,†,§ †

National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, Aškerčeva 5, SI-1000 Ljubljana, Slovenia § EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia ‡

S Supporting Information *

ABSTRACT: The structural properties of the mesoscopically confined drug and drug−drug and drug−matrix interactions were investigated in model drug-delivery systems prepared from nonfunctionalized and functionalized SBA-15 mesoporous silicate matrices, loaded with different amounts of indomethacin molecules. 1H MAS and 1H−13C CPMAS NMR spectroscopy indicated that only when the concentration of indomethacin within the mesopores becomes sufficiently high (when the mass fraction of indomethacin within the sample exceeds ∼0.15) do hydrogen bonds between the drug molecules become abundant. Nitrogen sorption analysis and comparison of 1H spin−lattice relaxation times in progressively loaded SBA-15 matrices suggested that at low loading concentrations indomethacin forms a layer on the silicate walls of the mesopores and that at moderate or high loading concentrations rigid nanoparticles that extend throughout the entire mesopore cross section are formed. 1H−29Si HETCOR NMR spectra indicated that the interaction between the indomethacin molecules and the silicate surface was moderate to weak. The 1H−13C CPMAS NMR spectrum of indomethacin embedded within the mesopores of SBA-15 closely resembled the spectrum of the bulk amorphous indomethacin and did not allow to draw firm conclusions about the molecular conformation and the packing of the drug molecules within the pores. On the contrary, variable-temperature 1H spin−lattice relaxation measurements showed that the mesoscopically confined indomethacin is significantly different from the bulk amorphous indomethacin. It does not become rubbery, and it exhibits a solid−solid transition at 363 K that is similar to the phase transition of the crystalline indomethacin solvate with tetrahydrofuran. When indomethacin is incorporated into the functionalized SBA-15 matrix, the interactions between the embedded drug molecules and the walls of the matrix are enhanced.

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INTRODUCTION Over the past years ordered mesoporous silicates have emerged as promising new materials for very diverse applications.1 They can be used as sensors,2,3 catalysts,4,5 and materials for chromatography;6 they are employed in wastewater treatment7 and also in nanomedicine and in pharmacy.8−10 Their wide applicability is mostly due to their large filling volumes, large surface areas, and very narrow pore-size distributions along with their high chemical and thermal stabilities.11−13 In the field of pharmacy they are especially important as the drug carriers that can accommodate drug molecules within their mesopores. Such drug-delivery systems successfully circumvent the problem of poor water solubility of hydrophobic drugs.14−17 On the internal and external surface, mesoporous silicates possess a significant amount of free silanol groups, which can be functionalized.18 The attached functional groups can change physicochemical properties of the surface and can therefore extend the area of possible applications of mesoporous silicates. For example, specific functional groups can lead to specific interactions of the pharmaceutical system with the target tissue. This makes functionalized mesoporous silicates also promising matrices for site-specific or/and stimuli-responsive drug delivery.19−24 © 2012 American Chemical Society

Detailed characterization of drug-delivery systems based on mesoporous silicates is vital for the understanding of the mechanisms of drug incorporation and release. A variety of techniques have been used so far, the most frequently employed being thermal analysis and nitrogen sorption, which can both give qualitative and quantitative information about bulk structural and thermodynamical properties of the incorporated drugs. Unfortunately, the two techniques do not provide any knowledge about the microscopic properties of the embedded drugs and in particular about the crucial drug−drug and drug−silicate interactions. X-ray powder diffraction (XRPD), which is the usual method of choice for the investigation of the atomic-level structural properties, is also inapplicable because of the lack of long-range order in phases confined to mesopores. The alternative technique is solid-state nuclear magnetic resonance (NMR), which through spectroscopy and relaxation measurements offers valuable information about structural and dynamical properties of the embedded compound and which is not limited to the materials or forms that exhibit long-range order.25−27 The technique even offers Received: September 9, 2011 Revised: January 4, 2012 Published: January 4, 2012 2662

dx.doi.org/10.1021/jp2087016 | J. Phys. Chem. C 2012, 116, 2662−2671

The Journal of Physical Chemistry C

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unreacted APTES. The sample was dried at 333 K for 6 h in ventilation dryer and for an additional 24 h in a vacuum dryer at 313 K. After drying the SBA-15/APTES sample was placed in a desiccator under an inert Ar atmosphere. The drug-loading procedure started with the preparation of solutions of indomethacin (IMC γ, Sigma) in tetrahydrofuran (THF). The solutions were added dropwise to fine layers of calcined SBA-15, allowing the powder to soak up the added drops. The obtained samples were then dried using a two-step drying procedure combining drying at 313 K for 24 h in a ventilation dryer and drying at 313 K for 24 h in a vacuum dryer. Immediately after drying, the samples were placed in a desiccator under inert Ar atmosphere to avoid exposure to moisture and were kept in such conditions until immediately before the measurements. Calcined SBA-15 was loaded with IMC solutions of three different concentrations (25 mg IMC/1 g THF, 50 mg IMC/1 g THF, and 150 mg IMC/1 g THF). The obtained composites were denoted as SBA-15/IMC (0.15), SBA-15/IMC (0.27), and SBA-15/IMC (0.50), where the numbers in the parentheses represent the mass fractions of IMC within the samples, m(IMC)/m(sample), as determined by the thermogravimetric analysis. The functionalized SBA-15/ APTES sample was loaded with the IMC solution with the concentration of 50 mg IMC/1 g THF. IMC γ was purchased from Sigma and used in the experiments as obtained. Form α was prepared from 5 g of IMC γ dissolved in 5 mL of ethanol at 80 °C. After 10 min, 30 mL of distilled water at room temperature was added to the IMC−ethanol solution at 80 °C. The precipitated crystals were removed by filtration and dried for 4 h at 50 °C. IMC β was prepared by recrystallization from THF. The described samples were inspected by X-ray powder diffraction, thermogravimetric techniques, and nitrogen sorption measurements. The experimental details along with the results of these analyses are presented in the Supporting Information. Solid-State Nuclear Magnetic Resonance. 1H and 29Si MAS and 1H−13C and 1H−29Si CPMAS NMR spectra were recorded on a 600 MHz Varian NMR System equipped with a 3.2 mm Varian MAS probehead. Larmor frequencies for 1H, 13 C, and 29Si nuclei were 599.714, 150.812, and 119.131 MHz, respectively. Chemical shifts of the three types of nuclei were reported relative to the signals of these nuclei in tetramethylsilane. Relaxation delays and sample rotation frequencies were 10 s and 20 kHz, respectively, for 1H MAS NMR, 30 s and 10 kHz for 29Si MAS NMR, and 5 s and 10 or 16 kHz for 1H−13C and 1 H−29Si CP MAS NMR measurements. CP contact times in the latter two experiments were 2.5 ms. Low-temperature CRAMPS (combined rotation and multiple-pulse sequence) measurements were conducted at 10 kHz sample spinning and employed supercycled windowed PMLG5 homonuclear decoupling scheme.39 For the decoupling the strength of the radiofrequency field was 96 kHz, the duration of the entire supercycle was 38.6 μs, and the duration of the sampling window was 3.3 μs. The sampling was performed after each of the two PMLG5 blocks within the supercycle. The twodimensional 1H−29Si HETCOR experiment was performed by first exciting the protons with a single pulse and letting the proton magnetization to evolve in the absence of homo- and heteronuclear decoupling; after that, the magnetization was transferred to silicon nuclei by a ramped cross-polarization block; during the evolution and acquisition of the silicon signal, high-power heteronuclear decoupling was applied. The two-

insight into the intermolecular or intramolecular interactions between the embedded molecules and between these molecules and the silica matrix.28 Solid-state NMR is also very powerful in the investigations of drug polymorphism.29 In this work we use solid-state NMR techniques to elucidate the microscopic properties of a model drug-delivery system. We use SBA-15 as a mesoporous silicate matrix and indomethacin as a model drug. SBA-15 is a well-known and widely investigated silicate with a hexagonal mesopore arrangement with a pore diameter usually between 5 and 10 nm. Indomethacin is a nonsteroidal anti-inflammatory drug, wellknown for its rich polymorphism.30−32 Three anhydrous forms of indomethacin are known. Form α is the metastable form, form β is the solvate or the so-called pseudopolymorph, and form γ is the stable form of indomethacin. The solvates can be formed from different solvents such as tetrahydrofuran, acetone, and dichloromethane.33 The structure and phase behavior of mesoscopically confined indomethacin were recently investigated by thermal analysis in combination with He pycnometry and NMR spectroscopy.34 One of the most important conclusions was that the phase diagram of indomethacin incorporated into the pores of SBA-15 with a pore diameter of around 10 nm comprises a disordered molecular dispersion and/or a metastable crystalline phase (β form). The molecular dispersion was formed from highly diluted loading solutions, and the metastable nanocrystalline phase was formed from moderately or highly concentrated loading solutions. It was also found that both phase formation and phase transformations of matter confined in nanodimensions differ significantly from those of the macroscopic crystalline phases. A simple thermodynamical argument has highlighted the importance of drug−surface interactions in the formation of the confined phase. To gain additional insight into the physical state of the mesoscopically confined indomethacin and to probe the drug−drug and drug−surface interactions, we employ here 1H, 13C, and 29Si magic-angle spinning NMR spectroscopy and 1H NMR spin−lattice relaxation measurements. We use two “types” of SBA-15: the as-prepared one and the one functionalized with 3-aminopropyltriethoxysilane (APTES) molecules.35 Between the two main approaches to surface functionalization, namely the in situ functionalization (co-condensation) and the postsynthetic functionalization (grafting), we used the latter, where one introduces new functional groups after the formation and calcination of mesopores without affecting the structural integrity and longrange periodicity of the final mesoporous material.36,37

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EXPERIMENTAL SECTION Preparation of Materials. Mesoporous SBA-15 silica was synthesized according to Sayari et al.38 using structure directing agent Pluronic P123 (PEG−PPG−PEG block copolymer, Aldrich) and tetraethyl orthosilicate (98% TEOS, Aldrich) as a silica source. Functionalization of SBA-15 was performed in a Schlenk line. First, 100 mg of calcined SBA-15 was outgassed under vacuum at 383 K for 2 h. After that, the samples were purged with flowing nitrogen for 15 min and then dispersed in 25 mL of dry toluene (Fluka, puriss., H2O ≤ 50 ppm) at ambient temperature under magnetic stirring. After the addition of a calculated amount of 3-triethoxysilylpropylamine (99% APTES, Sigma-Aldrich; 33 μL for the targeted functionalization of 100 mg of calcined SBA-15), the suspension was stirred for 24 h. The functionalized product was recovered by filtration with toluene. The filtration was repeated 5 times to remove the 2663



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Figure 1. 29Si MAS and 1H−29Si CPMAS NMR spectra of the calcined SBA-15 and of the SBA-15 functionalized by APTES molecules. Qn, Tn, and D2 label the signals assigned to different Si environments (for Qn and Tn see main text; D2 stands for R2Si(OSi)2). D2 belongs to an unidentified impurity.

the Q3 and Q2 signals in the 29Si MAS spectrum shows that about one-half of the silanol groups got involved in the bonds with APTES molecules. Considering the abundance of these molecules as deduced from the intensities of the T3 and T2 signals, it seems that in addition to the Si−O−Si bonds between the APTES molecules and the silicate surface some Si−O−Si bonds are formed also between the nearby APTES molecules themselves. Both SBA-15 materials were used as the drug-delivery matrices for the model molecule of IMC. A scheme of the IMC molecule, a 2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2methyl-1H-indol-3-yl}acetic acid, is presented in Figure 2. The substance forms two crystallographic polymorphs, a stable form γ,42 and a metastable form α43 as well as several solvates with various solvents.33 The molecular crystals of γ and α are formed as hydrogen-bonding networks of IMC molecules. In the hydrogen bonds carboxyl groups, hydrogen-bond donors and acceptors, and amide groups, hydrogen-bond acceptors, can be involved. Figure 3 shows that 1H−13C CPMAS and 1H MAS spectra of the two polymorphs of IMC and of the IMC solvate with THF provide a great deal of information on the structural properties of these solids. Namely, a simple inspection of the carbon spectra indicates that there is one IMC molecule in the asymmetric crystallographic unit of form γ (single resonances at, for example, 13.7, 28.4, and 55.4 ppm), two IMC molecules and a molecule of THF in the asymmetric unit of the solvate β (the resonances at about 13, 30, and 55 ppm are split into two peaks; the intensities of the signals at 26.1 and 68.2 ppm belonging to two THF −CH2− carbon atoms are approximately equal to the intensities of the signals at 29.3 and 30.7 ppm belonging to −CH2− carbon atoms in two molecules of IMC), and three molecules of IMC in the asymmetric crystallographic unit of form α (the resonances at about 13, 30, and 53 ppm are split into three peaks). Even more interesting is the information that is available from the proton

dimensional experiment was carried out in a hypercomplex mode.40 The number of increments along the indirectly detected dimension was 32, and the number of scans at each increment was 1200. 1H spin−spin relaxation and 1H spin− lattice relaxation were studied by Hahn-echo and inversion− recovery methods, respectively. The variable-temperature relaxation measurements were carried out with a 5 mm supersonic Doty probehead at MAS frequencies of 8 kHz. Repetition delays in relaxation measurements were approximately 5T1.

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RESULTS AND DISCUSSION Solid-state NMR spectroscopy, in particular 29Si MAS and 1 H−29Si CPMAS NMR spectroscopy, provides valuable information about the composition and structure of the mesoporous silicates. Figure 1 compares the spectra of the original, calcined SBA-15 material and of the material whose surface was functionalized by APTES molecules. The differences between the spectra are quite pronounced. The spectrum of the original material exhibits three overlapping contributions resonating at approximately −110, −100, and −90 ppm, which can be assigned to Q4 (Si(OSi)4), Q3 (Si(OSi)3(OH)), and Q2 (Si(OSi)2(OH)2) silicate species, respectively. Quantitative analysis of the spectrum shows that the material possesses ∼3.5 silanol groups per nm2 of the surface. The spectra of the functionalized material prove that the functionalization was successful. Not only the spectra of the functionalized material exhibit resonances at about −67 and −59 ppm, which can be assigned to T3 (RSi(OSi)3) and T2 (RSi(OSi)2(OH)) units,41 the relative intensities of the Q4, Q3, and Q2 signals are also substantially changed as compared to the original spectrum. The decrease of the density of “free” silanol groups unambiguously confirms that APTES molecules had attached to the surface. Quantitatively, the reduction of the intensities of 2664



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such an assignment was already done for IMC γ.44 The signal resonating at 179.1 ppm was assigned to the carboxyl carbon and the signal resonating at 167.3 ppm to the amide carbon. In our recent paper45 we have shown that using the known crystal structure of IMC γ, 1H and 13C isotropic chemical shifts can be also quite accurately predicted ab initio using the so-called GIPAW approach within the DFT frame.46 In the Supporting Information we show how this type of calculation has been extended to carry out the assignment of specific 1H and 13C NMR signals of IMC α. Because the detailed crystal structure of IMC β is not known, an equivalent assignment for this form could not be accomplished. The assignment of carbon resonances in the chemical shift range between 165 and 185 ppm and of proton resonances of hydrogen included in the hydrogen bonds is given in Figure 3. It is very interesting to note that isotropic chemical shifts of crystallographically inequivalent carboxyl carbon nuclei of IMC α can differ by as much as 10 ppm. While C1 and C2, which are both involved in a strong hydrogen bond, resonate very close one to another at 181 and 180 ppm, C3, which is involved in a weaker hydrogen bond, resonates at much smaller isotropic shift of 171 ppm. C1 and C2 are through oxygen connected to H1 and H2, which give rise to a proton NMR signal at 12.5 ppm, and C3 is connected to H3, which resonates at 11 ppm. The average O···H−O distance for the carboxyl−carboxyl type of bond involving H1 and H2 is 2.64 Å, and the O···H−O distance for the carboxyl−amide type of bond involving H3 is 2.74 Å. This observation suggests that the isotropic chemical shift of carboxyl carbon nuclei is very sensitive to the strength of the hydrogen bond. At weak bonds or, in the extreme limit, in the absence of them, the resonances of carboxyl carbon nuclei can closely approach the resonances of amide carbon nuclei. After using solid-state NMR spectroscopy to inspect the basic properties of the drug-delivery matrices and of the model drug itself, we can start the investigation of the incorporation of the drug into the mesoporous matrix. Figure 4 shows the 1 H−13C CPMAS spectra of the IMC molecules within the SBA15/IMC “composites”. All the spectra exhibit much broader signals than the spectra of the pure forms α, β, and γ. Clearly, carbon nuclei of IMC molecules embedded within the mesopores of SBA-15 experience a distribution of environments, which lead to a distribution of isotropic chemical shifts and further to broad but still quite well-resolved signals. For SBA-15/IMC composites with m(IMC)/m(sample) mass ratios of 0.15, 0.27, and 0.50 the 1H−13C CPMAS NMR spectra are substantially different. More than the difference in the signal strength, which reflects the quantity of IMC molecules within the sample, it is interesting to note that the appearance of the spectra in the frequency range between 165 and 185 ppm clearly deviates from one sample to another. For SBA-15/IMC (0.15) sample the peak at about 168 ppm is quite pronounced and stronger than the peak resonating at 156 ppm. Differently from that, for the composite SBA-15/IMC (0.50), the intensity of the former peak decreases, but new peaks between 170 and 180 ppm appear. Their widths are between 6 and 8 ppm. According to the conclusions of the previous paragraph, one reason for the differences between the spectra could stem from the differences in the bonding between the IMC molecules. In highly concentrated SBA-15/IMC samples the density of the IMC molecules within the pores is large, which is leading to close contacts and to establishment of hydrogen bonds among them. As opposed to that, in SBA-15/IMC samples with low

Figure 2. Chemical structure of IMC (2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl}acetic acid) (a) and hydrogen-bonding schemes between IMC molecules within the two polymorphs, γ and α (b). In (b) carboxyl carbon atoms are labeled by C1−C3, amide carbon atoms are labeled by A1−A3, and hydrogen atoms included in hydrogen bonds are labeled by H1−H3.

spectra. Proton signals corresponding to hydrogen nuclei that are involved in hydrogen bonds are clearly resolved from the rest of the proton signals and enable easy identification of the relative number of hydrogen atoms involved in the (different) hydrogen bonds. In the case of polymorph γ, a single resonance at about 12.8 ppm shows that in this form of IMC there is only one type of hydrogen bond, the bidentate bond between two carboxylic groups of the neighboring IMC molecules. Differently from that, in the case of the polymorph α, we can detect two signals that are corresponding to hydrogen-bonding protons. The quantification of the signals shows that per one asymmetric unit there are two protons involved in a bidentate bond between two carboxylic groups, thus in a similar bond to the one encountered in the form γ, and that there is also one proton involved in a hydrogen bond between a carboxylic and an amide group (Figure 2). Smaller isotropic chemical shift of the signal belonging to the latter proton indicates that the hydrogen bond involving a carboxylic and an amide group is weaker than the hydrogen bond between the two carboxylic groups. The 1H MAS NMR spectrum of IMC β exhibits only one signal belonging to hydrogen-bonded protons, thus indicating that there is only one type of hydrogen bond present in this form of IMC. For our later discussion it is useful to do a precise assignment of carbon NMR signals resonating between 165 and 185 ppm. Based on comparison with the solution 13C NMR spectrum, 2665



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Figure 3. 1H−13C CPMAS and 1H MAS NMR spectra of three forms of IMC. Asterisks in the carbon spectra denote the most intense spinningsidebands. Labels C1−C3, A1−A3, and H1−H3 denote carboxyl carbon nuclei, amide carbon nuclei, and hydrogen-bonded protons, respectively, to which particular NMR signals are assigned. The labels correspond to carbon and hydrogen sites as denoted in Figure 2.

the carbon spectrum of the corresponding sample. With the increased concentration of IMC within the pores of SBA-15, the fraction of THF becomes negligible. The same is true for the water molecules that give rise to a 1H NMR signal close to 5 ppm. In the sample with the smallest concentration of IMC the amount of water molecules is substantial. When the concentration of IMC is increased, the peak corresponding to water molecules vanishes. This is an indirect evidence that the pores become more and more filled with the drug molecules. Namely, the procedure of preparation of SBA-15/IMC samples, as described in the Experimental Section, is such that water molecules can enter the pores of SBA-15 only after the composite is already prepared. When transferred to the NMR rotors and waiting for the measurement, the materials are exposed to room atmosphere and can thus adsorb water from air. Proton spectra in Figure 5 show that the amount of the adsorbed water is substantial only in the material in which the concentration of IMC molecules is low, i.e., in the material in which the pores of the silicate matrix are not extensively filled with the drug. In part the decrease of the amount of water in the samples with the increasing amount of IMC should be attributed also to the increased hydrophobicity of the latter samples. In contrast to the spectra of pure forms of IMC, the roomtemperature proton spectra of SBA-15/IMC composites (Figure 4) do not exhibit any peaks that could be assigned to hydrogen-bonding protons. Only a closer look at the spectrum of the most concentrated material, SBA-15/IMC (0.50), reveals

concentration of the drug within the pores, contacts between the IMC molecules are rare. For very weak contacts (hydrogen bonds) and in the absence of them the amide and carboxyl carbon nuclei can resonate very close one to another and contribute to one broad and intense peak. When concentration increases, the broad peak at about 168 ppm splits into two distinct peaks: one at 169 ppm and another at 179 ppm. The former can be assigned to the amide carbon nuclei and the latter to the carboxyl carbon nuclei. The ratio of intensities of the signals at 168 and 156 ppm of 1.3 for SBA-15/IMC (0.15) is substantially larger than the ratio of intensities of the signals at 169 and 156 ppm of 0.9 for SBA15/IMC (0.50). This is another indication that in SBA-15/IMC (0.15) the broad signal at 168 ppm comprises contributions from both amide and carboxyl carbon nuclei. On the other hand, the intensity of the 168 ppm signal is nevertheless probably too small to suggest that all the carboxyl nuclei contributed to this signal. This means that because of the lack of the short-range order within SBA-15/IMC (0.15) the remaining carboxyl nuclei might give rise to a severely broadened signal, which in the spectrum with the poor signal-to-noise ratio becomes undetectable. Proton spectra of the differently concentrated SBA-15/IMC samples provide some additional information about the molecules within the mesopores. Narrow peaks at 1.1 and 3.6 ppm show that in the least concentrated sample, SBA-15/IMC (0.15), there is still some THF present. The peaks corresponding to THF molecules could also be observed in 2666



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Figure 4. 1H−13C CPMAS (a) and 1H MAS room-temperature (b) and low-temperature (c) NMR spectra of IMC within the differently concentrated samples of SBA-15/IMC. In the carbon spectra the dashed lines indicate the resonances of the carboxyl (179 ppm) and amide (169 ppm) carbon atoms within the most concentrated sample. In the proton spectra, the dashed line indicates the proton resonance of the water molecules and the dotted line circles the broad and weak contribution of protons included in the hydrogen bonds. Arrows point to the narrow peaks of THF. In (c) the spectra obtained by MAS at 10 kHz and by CRAMPS at 10 kHz are compared.

Figure 5. 1H−29Si HETCOR spectra of SBA-15/IMC (0.15) (a) and SBA-15/IMC (0.50) (b). For both samples the projection of the 2D spectrum on the 1H axis is compared to the room-temperature 1H MAS spectrum (plotted above the projection).

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mobile or are close to the pore walls and thus to the surface silanol groups. Because of the mobility of the IMC molecules and/or because of their coupling to silanols, the IMC protons are experiencing strong fluctuations of the dipolar interaction and are relaxing faster than the protons within the large IMC crystals of polymorphs α and γ. When the concentration of IMC is increasing, the pores of SBA-15 are getting filled and the molecules become more rigid. Also, they are no longer making just a layer at the pore wall but start to form particles that extend throughout the entire pore cross section. In such a particle not all the molecules are in contact with the silicate walls (and with the silanol groups). Consequently, because of the increased rigidity and/or because of the smaller fraction of the IMC molecules that are interacting with the pore surface, the average spin−lattice relaxation within the IMC particles becomes slower. In a way, spin−lattice relaxation times thus reflect the sizes and the numbers of the particles within the pores of SBA-15. The hypothesis about the formation of layers and particles is consistent also with the results of nitrogen sorption and X-ray diffraction measurements, which are presented in the Supporting Information. Another interesting information comes from 1H−29Si HETCOR spectroscopy. This type of spectroscopy exploits through-space dipolar couplings for the transfer of magnetization between 1H and 29Si nuclei and provides information about the proximity of these nuclei. As it can be seen in Figure 5, the spectra of SBA-15/IMC (0.15) and SBA-15/IMC (0.50) composites both exhibit cross-peaks among 29Si Q4, Q3, and Q2 signals and 1H signals that resonate between 0 and 8 ppm. Although the cross-peaks, i.e., the interactions, are stronger between silicon and the adsorbed water molecules (1H resonances between 4 and 5 ppm for SBA-15/IMC (0.15)) or the silanol groups (1H resonances between 2 and 4 ppm for SBA-15/IMC (0.50)), underneath these peaks there are nonnegligible broad contributions that can be assigned to protons of the IMC molecules. The spectra therefore indicate that there are weak or moderate interactions between the IMC molecules and the silicate surface. This also means that IMC molecules are close to the walls of SBA-15 in SBA-15/IMC (0.15) and that, unlike ibuprofen molecules in MCM-41,26 they are not mobile enough for the dipolar coupling to be entirely averaged out and for the transfer of magnetization to be suppressed. On the contrary, the absence of cross-peaks that could be assigned to THF molecules suggests that these latter molecules are very mobile. 1 H−13C CPMAS NMR spectra of SBA-15/IMC (0.27) and SBA-15/IMC (0.50) composites (see Figure 4) resemble closely the 1H−13C CPMAS NMR spectrum of the amorphous IMC.44 Literature describes several examples of crystalline substances that turn amorphous once they are confined within the mesopores.47,48 On the other hand, detailed calorimetric studies and measurements of the densities of the IMC particles embedded within the mesopores of SBA-15 showed that these particles exhibit properties that are typical for the (nano)crystals of IMC β.34 Therefore, it should be very interesting if some additional microscopic insight into the composition of the particles was obtained. Studies on nonconfined phases presented in refs 44 and 49 showed that important information about the physical state of the material can be extracted from the variable-temperature 1H NMR relaxation investigations. It was shown that at temperatures below ∼310 K amorphous IMC is relatively rigid and glassy. At glass-transition temperature of 316 K, IMC starts to transform into a more mobile,

a very low and broad hump in the frequency range between 11 and 14 ppm. However, when the latter sample is cooled down to 180 K and when in this way the motion and exchange of protons are suppressed, the signal belonging to hydrogenbonding protons becomes much more prominent, especially if MAS is accompanied by a more efficient homonuclear dipolar decoupling technique. As opposed to SBA-15/IMC (0.50), the low-temperature spectrum of the least concentrated sample SBA-15/IMC (0.15) still exhibits no detectable peak of the hydrogen-bonding protons. This shows that there are only few hydrogen bonds in this sample and that the signals of protons that are involved in hydrogen bonds are smeared over a broad chemical shift range. In conclusion, the results of 1H MAS and CRAMPS NMR spectroscopy thus complement the results of 1 H−13C CPMAS NMR spectroscopy and together support the hypothesis that when concentration of the drug within the pores is low, hydrogen bonds between the molecules are rare and that those bonds that might exist are very diverse in terms of bond angles and bond distances. On the contrary, in highly concentrated SBA-15/IMC composites the number, the strength, and the short-range order of hydrogen bonds drastically increase. Additional insight into structure and dynamics of the embedded molecules can be obtained by monitoring 1H spin−spin and spin−lattice relaxation. The results of the measurements on the three forms of IMC, and on the SBA-15/ IMC samples prepared with different concentrations of IMC, are collected in Table 1. With the increasing concentration of Table 1. Comparison of Spin-Lattice and Spin-Spin Relaxation Times in SBA-15/IMC Samples and in Crystalline Forms of IMCa sample IMC α IMC β IMC γ SBA-15/IMC (0.15) SBA-15/IMC (0.27) SBA-15/IMC (0.50)

T1 (s)

T2 (ms)

± ± ± ± ± ±

0.26 ± 0.01 0.22 ± 0.01 0.19 ± 0.01

2.7 3.3 3.2 1.0 1.2 2.1

0.2 0.2 0.2 0.1 0.1 0.1

a All values correspond to the 1H NMR peak resonating at ∼7 ppm. Measurements were carried out at room temperature under 20 kHz MAS.

IMC within SBA-15, the spin−spin relaxation times T2 are getting shorter, showing that homonuclear dipolar interaction among protons is getting stronger. This is a consequence of the reduced mobility and/or increased density of protons due to the increased concentration of IMC molecules within the pores. As opposed to the spin−spin relaxation, spin−lattice relaxation is getting slower as the concentration of IMC within the pores is increasing. Thus, the spin−lattice relaxation times T1 are the longest in the most concentrated SBA-15/IMC sample and in the pure crystalline polymorphs of IMC. These observations might be explained as follows. The dominant source of 1H spin−lattice relaxation are most probably fluctuations of the homonuclear 1H−1H dipolar interaction, which can be induced by the internal motions within the IMC molecules (rotations and vibrations) or by the motions of the surface silanol groups (vibrations of Si−OH and rotational diffusion of hydrogen around the silanol Si−O axis) to which protons of IMC can also be coupled. When concentration of IMC within the pores is small, the molecules are relatively 2668



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T1 dependence of the SBA-15/IMC sample exhibits no maximum corresponding to glass transition. (Also, 1H NMR spectra show no line narrowing within the entire temperature range.) The proton T1 of the embedded IMC is increasing throughout the inspected temperature range and exhibits a quite pronounced change in the slope at about 363 K. This temperature very well agrees with the temperature of the phase transition detected by the DSC measurements as described in ref 34. (One should recall here that the phase-transition temperature of the mesoscopically confined matter is usually different than the phase-transition temperature of the nonconfined bulk matter.) Though this 1H spin−lattice relaxation characteristic does not directly prove that the particles of IMC within the mesopores of SBA-15 are nanocrystals of the form β (as proposed in ref 34), it does show that the embedded IMC does not behave as the bulk amorphous IMC and that it does exhibit a sort of a phase transition. In this way it is similar to crystalline IMC β. So far the study focused on the incorporation of IMC into the “original”, nonfunctionalized SBA-15. In the last part of the paper we focus on the incorporation of the drug into the mesoporous silicate functionalized by APTES molecules. 1 H−13C CPMAS NMR spectra of the empty functionalized SBA-15, functionalized SBA-15 filled with IMC, and nonfunctionalized SBA-15/IMC (0.50) sample are compared one to another in Figure 7. The spectrum of the functionalized

rubbery material. By increasing the temperature even further, at 403 K crystallization into α form begins. While 1H T1 of the crystalline IMC α was monotonically increasing between 300 and 400 K, T1 of the bulk amorphous IMC exhibited a pronounced maximum at the glass-transition temperature. In Figure 6, we compare 1H NMR spin−lattice relaxation curves for crystalline forms α, β, and γ and for the SBA-15/IMC

Figure 6. Proton T1 data obtained at MAS with 8 kHz for bulk crystalline IMC forms α, β, and γ and for the SBA-15/IMC (0.27) composite. Straight dotted lines indicate the slopes of the selected curves.

(0.27) composite. Relaxation was studied in the temperature range between 303 and 423 K. While the values of T1 that are listed in Table 1 were measured at room temperature under MAS frequency of 20 kHz, the variable-temperature measurements were carried out using a different probe at a different sample rotation frequency of 8 kHz. MAS at such rotation frequency did not lead to well-resolved 1H NMR spectra. The relaxation times presented in Figure 6 were thus obtained by following the height of the highest peak in the poorly or nonresolved proton spectra. The relaxation curves obtained at individual temperatures were well fitted by monoexponential curves. As expected for the temperature range between 303 and 423 K, in crystalline IMC α and γ T1 is monotonically increasing. Differently from that, T1 of the crystalline IMC β exhibits a pronounced step at ∼388 K, a temperature of the desolvation and phase transition to IMC α. Of course, this remarkable change in spin−lattice relaxation time is irreversible. Upon decreasing the temperature, T1 of the transformed IMC thus roughly follows the T1 curve of IMC α, only that relaxationtime values in the former curve are slightly lower, which might be attributed to slightly poorer crystallinity of the material obtained in the NMR rotor. Even though the carbon MAS NMR spectrum of IMC within the pores of SBA-15 is almost identical to the carbon NMR spectrum of the bulk amorphous IMC, the T1 temperature dependence of the embedded IMC is quite different. First, the

Figure 7. Comparison of the 1H−13C CPMAS NMR spectra of the functionalized SBA-15 (top), functionalized SBA-15 filled with IMC (middle), and nonfunctionalized SBA-15 filled with IMC (bottom). Dashed lines indicate the positions of the resonances of the functional propylamine groups. Dotted lines indicate the positions of selected signals of IMC within the nonfunctionalized SBA-15. Two resonances in the spectrum of the functionalized SBA-15 filled with IMC are shifted (indicated by solid lines).

SBA-15 filled with IMC, SBA-15/APTES/IMC, clearly exhibits all the signals characteristic for both IMC molecules and the functional propylamine groups (of the APTES molecules) attached to the silicate surface. The comparison of the 2669



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intensities of individual signals belonging to the two sources enables only very rough quantitative estimation of the relative amounts of IMC with respect to the functional groups. Namely, cross-polarization experiment usually does not allow a quantitative analysis. Only if one compares signals of very similar groups, e.g., the signals of the CH2 groups in the propyl chain of the functional group and the signals of the CH2 groups in the molecule of IMC, a very rough estimation of their relative abundance can be obtained. Such a comparison of lines in the spectrum of the functionalized drug-delivery system shows that the number of IMC molecules within this material is roughly by a factor of ∼2 smaller than the number of functionalizing propylamine groups. A second interesting observation is related to the position of the resonances belonging to carboxyl carbon atoms and to the carbon atoms from the CH2 group attached to the carboxyl group. In the material with the functionalized drug-delivery matrix these two resonances of IMC are by about 2 ppm shifted with respect to the corresponding resonances of IMC in the nonfunctionalized matrix. We can again assign this shift to the influence of the bonding. It seems that IMC molecules in the functionalized SBA-15 form hydrogen bonds with the propylamine functional groups attached to the silicate surface, which are different (stronger) from the bonds between the two IMC molecules. Such bonding will have an influence on the kinetics of the drug release.

Article

ASSOCIATED CONTENT

S Supporting Information *

X-ray powder diffraction, thermogravimetric analyses, and nitrogen sorption measurements on samples of SBA-15/IMC; results of DFT/GIPAW calculations on IMC α and IMC γ. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ACKNOWLEDGMENTS This work was supported by the Slovenian Research Agency research program P1-0021. REFERENCES

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CONCLUSIONS Solid-state NMR spectroscopy and relaxation measurements provided valuable insight into the model drug-delivery system composed of IMC and SBA-15. At low concentration IMC molecules within the mesopores of SBA-15 form layers at the silicate walls. At moderate and high concentrations they start to form hydrogen bonds and condense into rigid particles that extend throughout the mesopore cross section. The absence and existence of hydrogen bonds are indirectly reflected in the room-temperature carbon MAS NMR spectra, more precisely in the NMR signals of the carboxyl and amide carbon nuclei, and in the low-temperature proton MAS NMR spectra. Although the carbon NMR spectra of highly concentrated IMC within the mesopores are practically identical to the NMR spectrum of the bulk amorphous IMC, the dynamic characteristics of the embedded indomethacin are significantly different from the characteristics of the bulk material. The mesoscopically confined IMC does not become rubbery and remains rigid in the entire temperature interval between 300 and 423 K. The embedded IMC rather resembles the crystalline IMC β because it exhibits a solid−solid phase transition at about 363 K. Functionalization of the SBA-15 matrix with APTES molecules enhances the interaction between the walls and the incorporated IMC molecules. Finally, the presented study suggests that the experimental approach based on solid-state NMR spectroscopy and relaxation investigations can provide the information about the structural properties of the drug embedded within the mesopores, and about the drug−drug and drug−matrix interactions also much more generally, for other drug-delivery systems. Such information is extremely important for the understanding of release kinetics and for the estimation of the total amount of the released drug. Information obtained with NMR spectroscopy is thus indispensable for a systematic design and tuning of controlled release systems in real-life applications. 2670



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