Confinement Effects on Drugs in Thermally Hydrocarbonized Porous

Article pubs.acs.org/Langmuir

Confinement Effects on Drugs in Thermally Hydrocarbonized Porous Silicon Ermei Mak̈ ila,̈ †,‡ Mónica P. A. Ferreira,‡ Henri Kivela,̈ § Sanna-Mari Niemi,† Alexandra Correia,‡ Mohammad-Ali Shahbazi,‡ Jussi Kauppila,§ Jouni Hirvonen,‡ Hélder A. Santos,‡ and Jarno Salonen*,† †

Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland § Laboratory of Materials Chemistry and Chemical Analysis, Department of Chemistry, University of Turku, FI-20014 Turku, Finland ‡

S Supporting Information *

ABSTRACT: Thermally hydrocarbonized porous silicon (THCPSi) microparticles were loaded with indomethacin (IMC) and griseofulvin (GSV) using three different payloads between 6.2−19.5 and 6.2−11.4 wt %, respectively. The drug loading parameters were selected to avoid crystallization of the drug molecules on the external surface of the particles that would block the pore entrances. The successfulness of the loadings was verified with TG, DSC, and XRPD measurements. The effects of the confinement of IMC and GSV into the small mesopores of THCPSi were analyzed with helium pycnometry, FTIR, and NMR spectroscopy. The results showed the density of the THCPSi loaded drugs to be ca. 10% lower than the bulk crystalline forms, while a melt quenched amorphous drugs showed a density reduction of 3− 7.5%. DSC and FTIR results confirmed that the drugs reside in an amorphous form within the THCPSi pores. Similar results were obtained with NMR, which also indicated that IMC may reside as both amorphous clusters and individual molecules within the pores. The 1H transverse relaxation times (T2) of amorphous and THCPSi loaded drugs showed IMC relaxation times of 0.28 ms for both the cases, whereas for GSV the values were 0.32 and 0.39 ms, respectively, indicating similar limited mobility in both cases. The results indicated that strong drug−carrier interactions were not necessary for stabilizing the amorphous state of the adsorbed drug. Dissolution tests using biorelevant media, fasted state simulated intestinal fluid (FaSSIF) and simulated gastric fluid (SGF), showed that THCPSi-loaded IMC and GSV were rapidly released in FaSSIF with comparable rates to the amorphous forms, whereas in SGF the THCPSi reduced the pH dependency in the dissolution of IMC. properties.10,11 However, the enhanced thermodynamic properties inherent to the amorphous state lead to a tendency of spontaneous crystallization into a more thermodynamically stable state, depending on the surrounding conditions during storage or dissolution.12,13 As recrystallization can effectively remove all the benefits obtained from amorphization, considerable interest has been placed on preventing crystallization from occurring when the delivery of an amorphous form drug is considered.14,15 The use of mesoporous inorganic carrier materials, such as porous silica and porous silicon (PSi), are becoming more common in current drug delivery applications.16−20 By adsorbing the drug molecules inside small mesopores with a diameter of roughly 10−20 times the molecular dimensions of the drug, crystallization becomes increasingly difficult as the pores do not provide enough space for nucleation, preventing recrystallization through physical confinement.21 Adsorption

1. INTRODUCTION Oral drug delivery can be considered as the easiest and most convenient route of drug administration. However, as the current trend of new drug molecules is moving toward lipophilic and poorly water-soluble large molecules due to the use of combinatorial chemistry and high throughput screening methods in drug development,1−3 the oral delivery route is becoming increasingly challenging. As the estimates show a growing number of new drug compounds suffering from poor bioavailability due to the low solubility and permeability,4 the need for multiple approaches aiming to improve dissolution and pharmacokinetic properties of these drugs is evident. Salt formation and cocrystallization are two common methods that may drastically improve the aqueous solubility of drugs,5−7 either alone or together with methods that reduce the crystal size and increase the surface-to-volume ratio of the dissolving particles.8 Another enticing approach to improve the solubility of drugs is by removal of the crystal structure through amorphization.9 In an amorphous state, the drug molecules have higher mobility and internal energy than their crystalline counterparts, resulting in highly enhanced aqueous dissolution © 2014 American Chemical Society

Received: November 6, 2013 Revised: January 7, 2014 Published: February 11, 2014 2196

dx.doi.org/10.1021/la404257m | Langmuir 2014, 30, 2196−2205

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The surface chemistry of the PSi microparticles was stabilized by thermal hydrocarbonization (THCPSi).30 In brief, the microparticles were initially immersed into HF−ethanol solution to refresh the surface hydrides, vacuum filtered, and dried at 65 °C. The refreshed, as-anodized PSi particles were then placed into a quartz tube under continuous N2 flow (1 L/min) at room temperature for 30 min to remove residual moisture and oxygen. Acetylene (C2H2) flow of 1 L/ min was added for 15 min at room temperature, after which the quartz tube was placed in a furnace at 500 °C for 15 min under N2/C2H2 flow. After the treatment, the tube was allowed to cool back to room temperature under N2 flow. 2.2. Drug Loading. The crystalline IMC was obtained from Hawkins Pharmaceuticals Inc. and GSV from Sigma-Aldrich. Amorphous IMC and GSV were prepared by heating the drugs slightly above their respective melting points (170 and 225 °C, respectively) for 10 min and rapidly quenching the melt with liquid N2. The drug loading into THCPSi was done by immersing the microparticles into loading solutions of different concentrations for 1 h under a gentle magnetic stirring, using a ratio of 160 mg of microparticles for 1 mL of the drug solution. For the loading, IMC was dissolved into dimethyl sulfoxide (DMSO) and GSV into dichloromethane (DCM). After loading, the particles were removed from the solution by vacuum filtration. The IMC loaded particles were dried in a vacuum oven at 105 °C for 72 h at a pressure of 12 mbar, while the GSV loaded particles were dried in a regular oven at 65 °C for 12 h. 2.3. Physical Characterization of the Samples. 2.3.1. Structural Characterization. The porous structures of the empty and drugloaded THCPSi microparticles were analyzed with N2 sorption at −196 °C (TriStar 3000, Micromeritics). The specific surface area was calculated from the adsorption isotherm using Brunauer−Emmett− Teller (BET) theory.37 The total pore volume was obtained from the amount adsorbed at relative pressure of p/p0 = 0.97.38 The average pore diameter was calculated from the obtained BET area and the pore volume by assuming the pore shape to be cylindrical. The pore size distribution was calculated from the desorption branch of the isotherm using Barrett−Joyner−Halenda (BJH) theory.39 The true densities of the samples were determined with helium pycnometry (AccuPyc 1330, Micromeritics) at room temperature using the average result of at least 10 repetitions. 2.3.2. Thermal Analysis. The drug payloads and the crystalline state of the drugs were studied with thermoanalytical methods. The total amount of the adsorbed drug was determined by thermogravimetry (TG; TGA-7, PerkinElmer) by evaporating the drug from the pores under continuous N2 flush of 200 mL/min. The temperature scan rate used was 40 °C/min. The weight loss of the empty particles, 0.4 ± 0.1 and 0.5 ± 0.1 wt %, for batches 1 and 2, respectively, were subtracted from the obtained payloads. Differential scanning calorimeter (DSC; Pyris Diamond DSC, PerkinElmer) was used to estimate the possible crystallization of the drugs loaded inside the THCPSi mesopores or on the surface of the microparticles. The crystallinity of the IMC and GSV reference samples was also analyzed with DSC. For the DSC measurements, the samples were sealed in 30 μL aluminum pans with holes in the lid. Measurements were performed with a scanning speed of 10 °C/min under continuous 40 mL/min N2 purge gas flow. The DSC sample amounts for crystalline and amorphous drugs were ca. 1.5 mg and for THCPSi-loaded samples ca. 2.5 mg. Both TG and DSC measurements were performed at least in duplicate. 2.3.3. X-ray Powder Diffraction. Complementary information on the crystallinity of the samples was acquired with X-ray powder diffraction (XRPD) measurements. The samples were measured using a Philips X’Pert Pro diffractometer with monochromated Cu Kα radiation. The voltage and current of the X-ray tube was set to 40 kV and 50 mA, respectively. The incident beam optics consisted of a 0.04 rad Soller slit with a 20 mm equatorial mask and 1/4° axial divergence slit, while the diffracted beam optics contained a 1/4° antiscatter slit followed by a 0.04 rad Soller slit and a programmable receiving slit before the proportional counter. The scans were made in a 2θ range of 5°−35° with a step size of 0.04° and a step time of 2 s using aluminum sample plates.

into the pores also lowers the Gibbs free energy of the drug molecules and may reduce their mobility, causing further hindrance for the crystalline phase formation.18,22 The applicability of a drug carrier material depends on the understanding how it affects the properties of the guest molecules. Several studies conducted on mesoporous silicas, such as MCM-41, SBA-15 and Sylysia, have shown that the drug molecules reside in an amorphous state in the mesopores, in some cases forming hydrogen bonds with the silanol- or amine-terminated silica walls.23−25 PSi shares several key features with the silicas, such as the dissolution enhancing properties by forcing the drug molecules into an amorphous state within the confined pores.26,27 The properties of PSi, however, offer several approaches that warrant further studies on the PSi-confined drug behavior. The native hydrogenterminated surface of PSi enables extensive control over the surface chemistry, ranging from oxidation28 to hydrosilylation29 and carbonization.30 The broad control enables, for example, simple multifunctionalization routes providing high surface coverage without significant structural alteration.31 The modification of the PSi surface chemistry by thermal hydrocarbonization with acetylene (THCPSi) provides a highly stable, hydrophobic surface for drug adsorption.30,32 The modification replaces the oxidation prone silicon hydrides with a hydrocarbon termination that has been shown to be relatively nontoxic33 and has been applied successfully in the delivery of small molecule drugs as well as peptides, enhancing their solubility and permeability characteristics.31,34,35 In contrast to oxidized PSi and porous silica, the THCPSi surface lacks surface groups such as silanols or other grafted functional moieties.27,36 As hydrogen bonding interactions between silica pore walls and the drug molecules have been observed and noted to affect the deposition of the drugs within the pores,23 THCPSi was selected as the carrier material for this study due to relatively inert nature of its surface. In this study, the physical states of two poorly water-soluble drugs, indomethacin (IMC) and griseofulvin (GSV), confined into the THCPSi mesopores were characterized comprehensively using several methods in order to elucidate the possible interactions taking place between the drug molecules and between the drugs and the THCPSi surface. The analyses were carried out using gas sorption, pycnometry, thermal analysis, and FTIR and NMR spectroscopic methods. The drug dissolution properties of the loaded THCPSi particles were also verified using physiologically relevant media FaSSIF (fasted state simulated intestinal fluid) and SGF (simulated gastric fluid) in sink and nonsink conditions.

2. EXPERIMENTAL SECTION 2.1. THCPSi Carrier Preparation. PSi microparticles were produced by electrochemically anodizing monocrystalline borondoped p+-type Si⟨100⟩ wafers (4 in.: Cemat Silicon S.A., Poland; 6 in. (batch 1): Okmetic Ltd., Espoo, Finland (batch 2)) with a resistivity of 0.01−0.02 Ω·cm in a 1:1 (vol) aqueous hydrofluoric acid (HF, 38%)−ethanol electrolyte using a constant current density of 50 mA/cm2. The porous layer was lifted off from the Si substrate by abruptly increasing the current density to the electropolishing region. The free-standing PSi films were then milled with a high energy ball mill (Pulverisette 7, Fritsch GmbH, Germany) in an agate grinding jar. The milled microparticles were dry and wet sieved with ethanol using test sieves. A size fraction of 53−75 μm was collected for the experiments. Scanning electron microscope images of the microparticles and the mesopore structure are provided in Figure S1 (Supporting Information). 2197


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2.3.4. Spectroscopic Analysis. Further information on the physical state of the samples was obtained with Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). The FTIR measurements were done using a Nicolet Nexus 870 FTIR spectrometer equipped with mercury−cadmium telluride detector cooled to −196 °C and a Harrick Seagull diffuse reflectance accessory. The sample chamber was continuously purged with a N2 gas flow. The samples were mixed into potassium bromide (1:20 w/w) and carefully ground with an agate mortar and pestle. Each spectrum was averaged over 256 scans. Solid-state 1H and 13C NMR spectra were measured with a Bruker Avance 400 spectrometer (1H: 399.75 MHz; 13C: 100.52 MHz), equipped with a 4 mm MAS (magic angle spinning) probe. Details of the experimental procedure are provided in Supporting Information 1. 2.3.5. Statistical Analysis. The statistical analysis of the results comparing the THCPSi-confined drug densities to the amorphous and crystalline references were carried out using a one-way analysis of variance (ANOVA), followed by a Games−Howell comparison test. The level of significance was set at a probability of *p < 0.1, **p < 0.05, and ***p < 0.01 between the THCPSi samples and the references and also between the amorphous and the crystalline references. Error bars represent the average standard deviations (n ≥ 3). The analyses were carried out using SPSS Statistics v22 software (IBM Corp.). 2.4. Drug Dissolution Studies. For the drug dissolution tests, FaSSIF and SGF were used as biorelevant media and prepared as described elsewhere.40 The dissolution tests were made in sink conditions by immersing the samples in FaSSIF and SGF at 37 °C using a stirring speed of 200 rpm. For the drug release quantification, 1 mL of the dissolution medium was withdrawn at each time point and then replaced with 1 mL of fresh, prewarmed media. The samples collected were centrifuged for 2 min and analyzed with HPLC in order to quantify the amount of drug released over time as described below. Furthermore, drug dissolution experiments at supersaturation were also conducted. The supersaturation experiments were made by placing samples containing a known excess of the drugs in FaSSIF and SGF at 37 °C and following the dissolution rate using a similar procedure as with the sink conditions experiments. The saturation concentrations of the drugs in FaSSIF and SGF were determined for IMC as 88 ± 9 and 2 ± 0.5 μg/mL and for GSV as 42 ± 4 and 21 ± 5 μg/mL, respectively. The purity and the amount of released IMC and GSV were analyzed using Agilent 1260 and Agilent 1100 HPLCs (Agilent Technologies), respectively. The sample injection volumes for analysis were 20 μL with both drugs. With IMC the HPLC mobile phase was composed of 0.2% H2PO4 (pH 2) and acetonitrile (MeCN) in a volumetric ratio of (35:65). The drug separation was achieved by using a Gemini C18 column (Gemini-NX, 3 μm C18, 110 Å; Phenomenex), with a flow rate of 1 mL/min monitoring the UV wavelength of 320 nm. For GSV, the mobile phase was composed of 0.1% trifluoroacetic acid (pH 2) and MeCN in a volumetric ratio of (45:55). The drug separation was done with a Gemini C18 column, with a flow rate of 1 mL/min at a UVwavelength of 292 nm.

Table 1. Specific Surface Area, Pore Volume, and Average Pore Diameter of the THCPSi Microparticle Batches Obtained from the N2 Sorptiona

THCPSi, batch 1 THCPSi, batch 2

specific surface area (m2/g)

total pore volume (cm3/g)

average pore diameter (nm)

true density (cm3/g)

232 ± 5

0.64 ± 0.01

11.1 ± 0.1

2.11 ± 0.01

261 ± 3

0.87 ± 0.01

13.3 ± 0.1

2.11 ± 0.01

a

Batch 1 was used exclusively with IMC, and batch 2 was used with GSV.

Table 2. Loading Solution Concentrations and Thermogravimetrically Determined Total Payloads q (wdrug/ wsample) of IMC and GSV in the THCPSi Microparticles

low medium high

IMC solution (mg/mL)

drug payload (wt %)

GSV solution (mg/mL)

drug payload (wt %)

125 250 400

6.2 ± 0.3 13.9 ± 0.2 19.5 ± 0.2

60 90 120

6.2 ± 0.3 9.8 ± 0.5 11.4 ± 0.3

the samples determined by TG. With IMC, the drug payload varied from roughly 6.2 to 19.5 wt %, while with GSV from 6.2 to 11.4 wt %, according to the loading solution concentration. Coarse visual determination of the saturation concentration showed IMC solubility into DMSO to be over 1000 mg/mL and GSV in DCM approximately 190 mg/mL at room temperature. The loading concentrations were then accordingly selected to be considerably lower than the drug saturation concentrations in the respective solvents. By using only low to moderate solution concentrations, the resulting payloads were similar, but the probability of drug crystallization on the external surface of the particles was clearly reduced.16 This was considered advantageous and preferable in characterizing the confinement effects on the drug molecules. The possibility of external surface crystallization of the drug molecules in porous materials can be evaluated using DSC measurements, which also allow the estimation of the amount of crystalline drug residing outside the pores.16 In addition, it is also possible to distinguish the crystallization of the drug within the confined space through the depression of the drug melting temperature from the bulk crystalline form according to the Gibbs−Thomson equation.42,43 This enables efficient preliminary characterization of the successfulness of the drug loading procedure. As is shown in Figure 1, the DSC results of the bulk crystalline and melt quenched, amorphous IMC, the crystalline γ-form of IMC shows a strong endotherm at 161 °C, in agreement with previous studies.44 The amorphous form, on the other hand, presents several events during temperature scan. Notably, a glass transition at 42 °C, as well as two weak exothermic events, were occurring at ca. 97 and 126 °C, which can be assigned to partial recrystallization. These are followed by a small melting endotherm at 159 °C, indicating that the sample was recrystallized back into γ-form. The GSV samples showed similar thermal behavior. The crystalline GSV provided a clear melting endotherm at 217 °C, while the amorphous sample showed a distinct glass transition at 89 °C followed by a strong exothermic recrystallization event starting at 128 °C, and finally a melting endotherm at 216 °C, consistent for partial recrystallization into the original form.

3. RESULTS AND DISCUSSION 3.1. Physical Characterization of the Drug-Loaded THCPSi Microparticles. The N2 sorption results show the two THCPSi batches to be structurally fairly similar, with the second batch appearing larger in respect of pore volume and surface area (Table 1). The obtained type IV isotherms are common for mesoporous silicon,38,41 with the BJH results showing both batches to have similar narrow pore size distributions, as shown in Figure S2. The true densities obtained for the two batches were also similar. Both of the THCPSi batches were used exclusively for one drug: batch 1 was utilized with IMC and batch 2 was used with GSV. The concentrations used in the loadings of IMC and GSV are listed in Table 2, along with the corresponding total drug payloads of 2198


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Figure 1. DSC curves for crystalline (i), melt quenched (ii), and THCPSi loaded with IMC (a) and GSV (b) with low (iii), medium (iv), or high (v) drug payload. Insets show the glass transition and recrystallization events occurring in the melt quenched samples.

Table 3. Measured Onset Temperatures and Heats of Fusion and Recrystallization Obtained from the Crystalline and Melt Quenched IMC and GSV indomethacin griseofulvin

crystalline, γ-form amorphous crystalline amorphous

Tfus [°C]

ΔHfus [J/g]

161 159 217 216

108 5.5 112 104

Tg [°C]

Trc [°C]

ΔHrc [J/g]

42

97/126

−0.4/−4.0

89

128

−79

Figure 2. X-ray diffractograms of the crystalline (i), amorphous (ii) and THCPSi with high (iii), medium (iv), and low (v) drug payload of IMC (a) and GSV (b).

Estimating the remaining crystalline content of the melt quenched IMC and GSV can be done by comparing the obtained enthalpies of fusion and recrystallization listed in Table 3 to the melting enthalpy of the bulk crystalline form. Assuming that the changes in the sample heat capacities after recrystallization are negligible, the crystalline fraction C, can be calculated as described in eq 1: C (%) =

a ΔHfus + ΔHrca × 100% c ΔHfus

sample fabrication and handling. As GSV molecules are quite rigid and have tendency to recrystallize easily due to their low conformational entropy,45 the partial crystallinity may be difficult to avoid in the rapid quenching process and the subsequent sample handling. To assess this possibility, XRPD measurements of the samples confirmed some of the observations of the DSC results. The crystalline IMC and GSV provided expected diffractograms,14,46 while the amorphous samples showed only the characteristic broad halo. This indicates that the degree of crystallinity detected by DSC with the melt quenched GSV may be overestimated (Figure 2). While the reference samples provide information where the main thermal events related to the drugs could occur, glass transition or partial recrystallization from IMC and GSV loaded THCPSi samples could not be discerned in the same temperature range The loaded samples did not present detectable drug melting occurring at or below the bulk melting

(1)

where ΔHafus and ΔHcfus are the heats of fusion for the melt quenched and crystalline samples, respectively, and ΔHarc is the recrystallization heat. The equation yields a crystallinity of ∼2% for the IMC and ∼22% for the GSV. The values show the melt quenching of the IMC to have succeeded fairly well, while the GSV samples appear to have recrystallized partially during the 2199


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Figure 3. True densities of the THCPSi loaded microparticles and bulk forms of IMC (a) and GSV (b). Statistical analysis was made by ANOVA. The level of significance was set at probabilities of *p < 0.1, **p < 0.05, and ***p < 0.01 between the loaded microparticles and the amorphous/ crystalline sample. Errors bars represent the average ± SD (n ≥ 3).

where q is the drug payload (wdrug/wsample) and ρPSi, ρsample, and ρloaded are the true densities of the empty PSi microparticles, the loaded microparticles, and the loaded drug, respectively. Rearranging eq 2 for ρloaded yields qρPSi × ρsample ρloaded = qρsample + ρPSi − ρsample (3)

points of IMC and GSV, thus excluding also recrystallization to other polymorphs. Also, neither IMC nor GSV loaded particles showed broad endotherms encompassing wide temperature areas well below their crystalline melting points. This indicates that the size of the pores may be sufficiently small to prevent the formation of a long-range molecular ordering, leaving the drug into disordered, amorphous state. Similarly, the diffractograms of the loaded samples present only the Si⟨111⟩ peak at 28.4° due to the crystalline nature of PSi. The stability of the amorphous state was verified by placing the samples into stressed storage conditions at 40 °C and 75% relative humidity. The melt quenched samples appeared to crystallize almost completely after 7 days, while the THCPSi loaded samples did not show differences in their respective DSC results after 4 months of storage, as shown in Figures S3 and S4. The storage conditions presumably would have triggered the crystallization of any amorphous drug left on the external surface of the particles as well as within the pores. The results thus confirm that careful selection of the loading parameters was successful and that the THCPSi can be considered as a stabilizing matrix for the amorphous state of the drugs. The lack of detectable features in the DSC curves, however, hindered a more detailed analysis on the solid state structures of the drugs within the physical confinement of the pores. Formation of a partially crystalline structure within the pores could be used to characterize the interaction of the drug molecules with the pore walls by analyzing the thickness of the δ-layer, the disordered layer formed between the pore wall surface and the nucleated crystallites.43 However, the avoidance of surface crystallization, i.e., the possible pore blocking, is essential when considering the determination of the true density of the drug within the confined space with helium pycnometry. In pycnometry, the small sized helium molecules may enter to the rather small, partially filled pores and provide additional information about the solid state structure of the confined drug. Pycnometry can be used to estimate the drug load by utilizing the measured difference between the true densities of empty and loaded PSi with eq 2:

q=

With amorphous materials, the lack of long-range order between the molecules leads to the increase of its free volume due to the irregular packing. This is observable from the reduction in the densities of amorphous IMC and GSV shown in Figure 3, as the density of the drugs is reduced by ca. 3% and 7.5%, respectively, compared to the crystalline forms. The density of IMC in melt quenched amorphous form is known to vary from 1.31 to 1.35 g/cm3 depending on the cooling rate employed.47 As γ-form IMC has slightly lower density compared to the α-form (1.40 g/cm3), where the IMC molecules form trimers instead of dimers, it explains partially the limited difference between the bulk crystalline and the amorphous forms. While the DSC results indicated that the melt quenched GSV possibly retained a degree of crystallinity, the effect of this fraction does not appear to affect the obtained results. The density of the amorphous GSV sample is considerably lower compared to the crystalline form and close to the value obtained elsewhere for melt quenched GSV.48 Confining the drug to the mesopores shows a further reduction in their density, as the difference to the bulk crystalline form increased to ∼10% with both IMC and GSV. A notable feature with both the drugs is that the respective true densities of the confined drugs seem to be fairly similar despite the markedly different drug payloads. This indicates that there should not be considerable blockage for helium entry into the pores, which could cause underestimation in the density. Nitrogen sorption measurements for the loaded samples support this assumption, as the available pore volume is reduced in proportion to the drug payload (Figure S5 and Table S1). The further reduction in density could be partially explained by how the drug is dispersed within the pores. With mesoporous silicas, some drugs have been shown to reside both as individual molecules and clusters inside the pores.24,49 This increases the loss of order compared to the bulk

ρPSi − ρsample ρPSi − ρloaded

(2) 2200


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Figure 4. FTIR spectra of crystalline (i), amorphous (ii), and THCPSi with high (iii), medium (iv), and low payload (v) of IMC (a) and GSV (b). The spectra have been vertically shifted for clarity.

carboxylic acid dimer at 1718 cm−1 and to the benzoyl CO stretching at 1692 cm−1 (full spectra available in Figure S8).53 Amorphization of the compound causes several notable changes in the FTIR spectrum. The carbonyl-stretch-related bands show considerable broadening, as the state of the drug changes from crystalline to amorphous. The ν(CO) band related to the cyclic dimer shifts to 1710 cm−1, while a clear shoulder structure appears at 1735 cm−1, indicating the presence of non-hydrogen-bonded IMC molecules. The benzoyl related CO stretch also undergoes a slight shift to 1684 cm−1 due to the loss of order, while the CC stretching band at 1605 cm−1 cannot be observed after the amorphization. Comparing the amorphous IMC spectrum to the THCPSi confined form, the spectra appear quite similar, with the IMC related features becoming easier to resolve with increasing payload. The spectrum retains its amorphous-like appearance with broadened bands, and the presence of both hydrogenbonded and non-hydrogen-bonded CO peaks. The similarity of the spectra does not provide reliable insight on the amount of possibly clustered IMC in comparison to the bulk amorphous form. The crystalline GSV sample has also several CO related stretching bands. The bands at 1710 and 1660 cm−1 can be assigned to the carbonyl groups attached to the benzofuran and cyclohexene structures, respectively.54 After melt quenching of the GSV, the spectrum shows only limited changes. The differences are mainly observable as broadening of the CO related bands and the disappearance of the CC stretching band at 1605 cm−1 as with the IMC. Since the THCPSi provides no hydrogen bond donors, further differences compared to the spectrum of the amorphous GSV are not observed from the loaded samples. With both IMC and GSV, the FTIR results indicate that there is no observable interaction between the THCPSi surface and the drug molecules. The hydrophobic nature of the THCPSi however cannot be ruled out, which could lead to some form of hydrophobic interaction between the pore walls and the drugs. To gain further information on the amorphous nature and possible interactions affecting the adsorbed drugs, solid-state NMR measurements were performed on the high payload IMC sample (drug/sample ∼20%) and the medium payload GSV sample (drug/sample ∼10%) comparing these to the crystalline and amorphous forms of the drugs.

amorphous state, where short-range order is still present between the drug molecules. This possible presence of amorphous clusters with a number of individual molecules provides a plausible explanation for the observed reduced density. The method of loading the drugs using solvent immersion also supports this assumption of partial clustering within the mesopores.24 3.2. Spectroscopic Characterization of the Confined Drugs in THCPSi Microparticles. Possible interactions between the THCPSi surface and the loaded drugs were studied with FTIR. While the interactions between the pore walls and the drug molecules effect on the drug loading,23 their possible role in disrupting the crystalline state formation inside the pores remains still an open question. IMC and GSV differ in their respective crystalline forms from each other. IMC tends to form cyclic dimer structures through hydrogen bonds between the carboxylic acid groups. IMC also contains several hydrogen bond acceptor and donor sites that allow for several possible interactions with the porous structures, such as with silanol terminated and amine-terminated porous silica.50 GSV, on the other hand, has only hydrogen bond acceptor sites, preventing dimerization, but the presence of acceptor sites would not exclude possible interactions with surface silanols (Figure S6). Thermal hydrocarbonization of the PSi covers the surfaces with hydrocarbon species, maintaining the hydrophobic nature of fresh PSi. The FTIR spectrum of the THCPSi microparticles presented in Figure S7 shows various bands related to symmetric and asymmetric CH2 and CH3 stretching vibrations at 2850−2970 cm−1 with a terminal vinyl group band at 3050 cm−1. The broad band around 1720 cm−1 can be ascribed to stretching vibrations of acyloxy groups (ν(CO)). At lower wavenumbers (1200−1600 cm−1) the multiple bands can be assigned to different bending vibrations of CH2 and CH3 groups. In this respect, the hydrophobic THCPSi surface chemistry is considerably different from the hydrophilic silica or oxidized PSi structures used previously with IMC and GSV.27,51,52 In the FTIR spectra, the region between 1500 and 1800 cm−1 contains the bands related to the different CO and CC vibrations present in the IMC and GSV, allowing the evaluation of possible interactions between the drug molecules and the pore walls. The crystalline IMC spectrum shown in Figure 4 presents strong bands related to the CO stretching of the 2201


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Figure 5. 13C CP/MAS solid state NMR spectra of crystalline (i), amorphous (ii), and THCPSi (iii) loaded IMC (a) and GSV (b) at νMAS = 12 kHz.

areas from 120 to 150 ppm and from 5 to 50 ppm, which are related to the various hydrocarbon structures, such as vinyl and alkyl groups covering the THCPSi external surface, respectively. The strong background caused by the THCPSi, however, did not appear to affect the GSV resonances, as shown in the calculated difference spectrum in Figure S9. The deshielding of the carbonyl structures show that the local environment of GSV molecule is altered after the confinement, but its extent compared to the bulk amorphous form appears limited. This lack of changes confirms that GSV does not have strong interactions with the THCPSi as it was expected. Possible clustering of GSV molecules within the mesopores could be presumed according to the loading method used, but similar verification as with IMC was not obtained. 1 H MAS spectra of the confined samples were collected to obtain more information on the molecular mobility of the drugs within the pores. With ibuprofen and benzoic acid adsorbed in MCM-41, long transverse relaxation times T2 of 15−30 ms have been observed, accompanied by liquid-like narrow spectra indicating high drug mobility within the pores.55,56 Conversely, with itraconazole and IMC loaded in SBA-15, the mobility has been considerably lower, with obtained T2 times between 0.2 and 0.3 ms.50,57 The broad features presented in Figure S10 indicate short relaxation times, which the determinations confirmed. With amorphous IMC a T2 relaxation time ca. 0.28 ms was obtained, whereas with GSV the T2 time was ca. 0.32 ms. With the THCPSi confined samples, the relaxation times did not show notable change, as the obtained values were 0.28 and 0.39 ms for IMC and GSV, respectively. No previous relaxation times for confined GSV have been reported; however, the IMC value is comparable with confinement into SBA-15.50 Both results, however, point to limited mobility of the drug molecules inside the pores, comparable only to that of the bulk amorphous state. 3.3. Drug Dissolution Studies of Loaded THCPSi Microparticles. The dissolution behavior of IMC and GSV from THCPSi microparticles was studied using two physiologically relevant media, FaSSIF and SGF, in order to compare the drug release profiles of the confined drug loaded THCPSi samples to the crystalline and amorphous references. For these studies, the high payload sample of IMC (∼20 wt %) and medium payload sample of GSV (∼10 wt %) were selected. THCPSi has been previously shown as an effective drug carrier material, enhancing the drug dissolution and permeation of poorly water-soluble drugs as well as small peptides.31,34 In

As the FTIR spectra of the THCPSi loaded and the amorphous drugs presented very little differences, NMR provides further information about the immediate surroundings of the drug molecules. Figure 5a presents the solid state 13C CP/MAS spectra of the IMC samples. Compared to the crystalline γ-form IMC spectra, the bulk and confined amorphous spectra show considerable peak broadening due to the loss of long-range order, causing the molecules in different environments to display slightly different chemical shifts. The amorphous IMC shows, along with the typical peak broadening, a shift in the carboxyl carbon from 179.2 to 178.8 ppm, due to the presence of non-hydrogen-bonded IMC molecules. This resonance is shifted further to 178.1 ppm in the THCPSi confined form. The benzoyl carbonyl resonance at 167.7 ppm experiences also slight shifts after the amorphization to 168.1 ppm, showing increasing deshielding around the functional group. Confinement into THCPSi shifts the resonance peak to 168.2 ppm, indicating only a slight change from the bulk amorphous form. The amorphous and THCPSi confined IMC present both two additional peaks between the carboxyl and carbonyl carbon resonances. While the effect of the background noise in the THCPSi confined IMC signal due to the low overall amount of drug cannot be completely ruled out, the shoulder structures appearing at ca. 172 and 174 ppm are possibly related to different populations of non-hydrogen-bonded IMC present in amorphous clusters and individual molecules. The 13C spectra of GSV shown in Figure 5b mirror the results obtained with FTIR (Figure 4). The transformation from the crystalline to amorphous state broadens the peaks as expected, with the most notable changes taking place around the GSV methoxy groups, where the resonance peaks caused by the three −OMe moieties at 55.6, 58.9, and 59.8 ppm convolute into a single broad resonance at 56.8 ppm after the amorphization. The benzofuran and cyclohexene carbonyl resonances appear to undergo a degree of deshielding, with the latter showing a shift from 190.8 ppm in crystalline state to 192.1 ppm in amorphous and 191.8 ppm in THCPSi confined state and the former a stepwise 0.5 ppm shift starting from 195.1 ppm in the crystalline state and shifting to 195.6 and 196.1 ppm in amorphous and THCPSi confined states, respectively. As the THCPSi samples contains only fairly small amount of GSV, the 13C spectrum of the THCPSi becomes more prominent in the signal. This is shown as broad resonance 2202


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Figure 6. Drug dissolution profiles of crystalline, amorphous and THCPSi loaded IMC (a, b) and GSV (c, d) in FaSSIF (a, c) and SGF (b, d) under sink conditions at 37 °C. The amounts of available drug for dissolution were set to 2.5 (a), 3.5 (b), 1.1 (c), and 1.1 μg/mL (d).

As GSV is a poorly soluble, nonionizable molecule, the release rate was presumed to be pH independent. Comparing the crystalline and amorphous GSV release profiles in both FaSSIF and SGF, the dissolution behavior appears fairly similar, with the amorphous form dissolving slightly faster due to its higher free energy. In THCPSi microparticles, the drug release into both media is rapid, but the released amount during the observed 120 min remains consistently lower in SGF than in FaSSIF media. Overall, the release behavior of THCPSi loaded with GSV and IMC shows the combined effectiveness of amorphous state delivery to the large surface area of the particles available for dissolution. The use of biorelevant FaSSIF and SGF media is also interesting when considering the solubilizing properties of the buffers. Figure S11 shows that FaSSIF can solubilize IMC rather well regardless of the state of the drug. In contrast to IMC, the dissolution of the THCPSi-loaded GSV samples indicated considerable improvement over the crystalline and amorphous samples. In FaSSIF, the THCPSi-loaded GSV is completely released after 8 h, while the amorphous and crystalline samples reach only the saturation level in the same time frame. However, in SGF, while THCPSi-loaded GSV initially presents higher concentration than the two reference samples, all three samples show some degree of precipitation taking place after 8 h of dissolution experiments.

some cases, the hydrophobic nature of THCPSi may hinder effective dissolution, prompting the development of suitable coating methods to alleviate this tendency.35 In these experiments, the THCPSi samples were used bare, relying on the solubilizing effect of the amphipathic bile salts containing buffers that act as surfactants, enhancing the wetting of the particles and diffuse into the pores replacing the drug molecules. The dissolution profiles of IMC in FaSSIF and SGF under sink conditions are shown in Figure 6. In FaSSIF, the amorphous IMC appears to dissolve rapidly, with over 60% of the drug dissolved in the first 10 min, while the THCPSi-loaded and crystalline IMC release the drug slightly slower. The differences between free drug forms and the drug-loaded THCPSi however become evident after 90 min, where the THCPSi confined drug has been nearly completely released, while the dissolution of the free IMC forms a plateau below 80%. As IMC is a weak acid with a pKa of 4.5, its solubility in SGF is considerably lower than at higher pH conditions. This is also reflected in the dissolution rate; after the initial burst during the first 10 min showing ∼3% release, the dissolution continues at almost zero-order fashion, reaching 5% release after 120 min. The amorphous IMC shows initially slightly faster dissolution but levels also after the first 10 min, showing only slow release over the observed time period. Previous studies have shown a reduction in the pH-dependent dissolution with PSi,58 which is also observable in this case, as the IMC continues to release steadily from the THCPSi microparticles after the initial burst.

4. CONCLUSIONS The use of inorganic PSi materials with different surface chemistries is becoming more commonly studied, due to the 2203


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Solubility and Dissolution Rates. Adv. Drug Delivery Rev. 2007, 59, 617−30. (7) Elder, D. P.; Holm, R.; Diego, H. L. De Use of Pharmaceutical Salts and Cocrystals to Address the Issue of Poor Solubility. Int. J. Pharm. 2013, 453, 88−100. (8) Rasenack, N.; Müller, B. W. Dissolution Rate Enhancement by in Situ Micronization of Poorly Water-Soluble Drugs. Pharm. Res. 2002, 19, 1894−900. (9) Hancock, B. C.; Parks, M. What Is the True Solubility Advantage for Amorphous Pharmaceuticals? Pharm. Res. 2000, 17, 397−404. (10) Hancock, B. C.; Zografi, G. Characteristics and Significance of the Amorphous State in Pharmaceutical Systems. J. Pharm. Sci. 1997, 86, 1−12. (11) Yu, L. Amorphous Pharmaceutical Solids: Preparation, Characterization and Stabilization. Adv. Drug Delivery Rev. 2001, 48, 27−42. (12) Greco, K.; Bogner, R. Crystallization of Amorphous Indomethacin During Dissolution: Effect of Processing and Annealing. Mol. Pharmaceutics 2010, 7, 1406−18. (13) Ahmed, H.; Buckton, G.; Rawlins, D. A. Crystallisation of Partially Amorphous Griseofulvin in Water Vapour: Determination of Kinetic Parameters Using Isothermal Heat Conduction Microcalorimetry. Int. J. Pharm. 1998, 167, 139−145. (14) Löbmann, K.; Laitinen, R.; Grohganz, H.; Gordon, K. C.; Strachan, C.; Rades, T. Coamorphous Drug Systems: Enhanced Physical Stability and Dissolution Rate of Indomethacin and Naproxen. Mol. Pharmaceutics 2011, 8, 1919−28. (15) Laitinen, R.; Löbmann, K.; Strachan, C. J.; Grohganz, H.; Rades, T. Emerging Trends in the Stabilization of Amorphous Drugs. Int. J. Pharm. 2013, 453, 65−79. (16) Salonen, J.; Kaukonen, A. M.; Hirvonen, J.; Lehto, V. P. Mesoporous Silicon in Drug Delivery Applications. J. Pharm. Sci. 2008, 97, 632−653. (17) Manzano, M.; Colilla, M.; Vallet-Regí, M. Drug Delivery from Ordered Mesoporous Matrices. Expert Opin. Drug Delivery 2009, 6, 1383−400. (18) Qian, K. K.; Bogner, R. H. Application of Mesoporous Silicon Dioxide and Silicate in Oral Amorphous Drug Delivery Systems. J. Pharm. Sci. 2012, 101, 444−63. (19) Anglin, E. J.; Cheng, L.; Freeman, W. R.; Sailor, M. J. Porous Silicon in Drug Delivery Devices and Materials. Adv. Drug Delivery Rev. 2008, 60, 1266−77. (20) Prestidge, C. A.; Barnes, T. J.; Lau, C.-H.; Barnett, C.; Loni, A.; Canham, L. Mesoporous Silicon: a Platform for the Delivery of Therapeutics. Expert Opin. Drug Delivery 2007, 4, 101−10. (21) Alba-Simionesco, C.; Coasne, B.; Dosseh, G.; Dudziak, G.; Gubbins, K. E.; Radhakrishnan, R.; Sliwinska-Bartkowiak, M. Effects of Confinement on Freezing and Melting. J. Phys.: Condens. Matter 2006, 18, R15−R68. (22) Lyklema, J. Adsorption at Solid−liquid Interfaces with Special Reference to Emulsion Systems. Colloids Surf., A 1994, 91, 25−38. (23) Nieto, A.; Colilla, M.; Balas, F.; Vallet-Regí, M. Surface Electrochemistry of Mesoporous Silicas as a Key Factor in the Design of Tailored Delivery Devices. Langmuir 2010, 26, 5038−49. (24) Mellaerts, R.; Jammaer, J. A. G.; Van Speybroeck, M.; Chen, H.; Van Humbeeck, J.; Augustijns, P.; Van den Mooter, G.; Martens, J. A. Physical State of Poorly Water Soluble Therapeutic Molecules Loaded into SBA-15 Ordered Mesoporous Silica Carriers: a Case Study with Itraconazole and Ibuprofen. Langmuir 2008, 24, 8651−9. (25) Vogt, F. G.; Roberts-Skilton, K.; Kennedy-Gabb, S. A. A SolidState NMR Study of Amorphous Ezetimibe Dispersions in Mesoporous Silica. Pharm. Res. 2013, 30, 2315−31. (26) Salonen, J.; Laitinen, L.; Kaukonen, A. M.; Tuura, J.; Björkqvist, M.; Heikkilä, T.; Vähä-Heikkilä, K.; Hirvonen, J.; Lehto, V.-P. Mesoporous Silicon Microparticles for Oral Drug Delivery: Loading and Release of Five Model Drugs. J. Controlled Release 2005, 108, 362−74.

growing need for stabilizing matrices for amorphous state drug delivery. This work shows the effects of confining two small drug molecules caused by hydrophobic mesoporous THCPSi microparticles, focusing on elucidating the necessity of the carrier−drug interactions in stabilizing the amorphous state of the adsorbed drug. Carefully selected loading conditions enabled the determination of the true density of the confined drug molecules within the mesopores, showing a reduction compared to normal amorphous state. Spectroscopic analysis confirmed the amorphous nature of the drug molecules inside the pores and did not show any clear indications of interactions between the pore walls and the drug taking place. Combined with the reduced density results, the NMR spectroscopic analysis indicates the drug to reside possibly as amorphous clusters inside the pores. Thermal analysis also indicated the THCPSi to be able to stabilize the amorphous state for at least 4 months in stressed storage conditions.

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ASSOCIATED CONTENT

* Supporting Information S

SEM images; NMR experimental details; N2 sorption isotherms and pore size distribution of empty THCPSi; DSC scans of stressed samples; N2 sorption isotherms of loaded samples; chemical structures of IMC and GSV; full FTIR spectra of all samples; NMR difference spectra of GSV-loaded sample; 1H MAS spectra of IMC and GSV samples; dissolution profiles of IMC and GSV samples in nonsink conditions. 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: jarno.salonen@utu.fi (J.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. H. A. Santos acknowledges financial support from the Academy of Finland (decision nos. 252215 and 256394), the University of Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007−2013) grant no. 310892. Dr. Marianna Kemell, University of Helsinki, is acknowledged for providing the SEM images.

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REFERENCES

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