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Crystallization of Probucol in Nanoparticles Revealed by AFM Analysis in Aqueous Solution Kiichi Egami,† Kenjirou Higashi,† Keiji Yamamoto, and Kunikazu Moribe* Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan S Supporting Information *

ABSTRACT: The crystallization behavior of a pharmaceutical drug in nanoparticles was directly evaluated by atomic force microscopy (AFM) force curve measurements in aqueous solution. A ternary spray-dried sample (SPD) was prepared by spray drying the organic solvent containing probucol (PBC), hypromellose (HPMC), and sodium dodecyl sulfate (SDS). The amorphization of PBC in the ternary SPD was confirmed by powder X-ray diffraction (PXRD) and solid-state 13C NMR measurements. A nanosuspension containing quite small particles of 25 nm in size was successfully prepared immediately after dispersion of the ternary SPD into water. Furthermore, solution-state 1H NMR measurements revealed that a portion of HPMC coexisted with PBC as a mixed state in the freshly prepared nanosuspension particles. After storing the nanosuspension at 25 °C, a gradual increase in the size of the nanoparticles was observed, and the particle size changed to 93.9 nm after 7 days. AFM enabled the direct observation of the morphology and agglomeration behavior of the nanoparticles in water. Moreover, AFM force−distance curves were changed from (I) to (IV), depending on the storage period, as follows: (I) complete indentation within an applied force of 1 nN, (II) complete indentation with an applied force of 1−5 nN, (III) partial indentation with an applied force of 5 nN, and (IV) nearly no indentation with an applied force of 5 nN. This stiffness increase of the nanoparticles was attributed to gradual changes in the molecular state of PBC from the amorphous to the crystal state. Solid-state 13C NMR measurements of the freeze-dried samples demonstrated the presence of metastable PBC Form II crystals in the stored nanosuspension, strongly supporting the AFM results. KEYWORDS: atomic force microscopy (AFM), nanosuspension, agglomeration, crystallization, NMR spectroscopy

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INTRODUCTION In recent years, many novel drug candidates have displayed properties of poor water solubility and low membrane permeability due to their having a high molecular weight. Therefore, improving these pharmaceutical characteristics is a crucial issue before marketing a product. The biopharmaceutical classification system (BCS) categorizes drugs into one of four biopharmaceutical classes according to their aqueous solubility and membrane permeability characteristics.1,2 The drug molecules showing high membrane permeability but low solubility are BCS-classified into class II. The bioavailability enhancement of these compounds could be achieved by changing their physical properties using pharmaceutical techniques. Nanoparticles have attracted particular attention as a representative formulation strategy to improve the solubility of poorly water-soluble drugs.3−5 Reducing the size of a drug below the submicron order has been achieved utilizing the grinding technique,6−8 the precipitation method,9,10 and the homogenization approach.11,12 Nanosizing of drugs provides significant increases in the specific surface area, resulting in an improvement of the dissolution rate. Furthermore, an increase © 2015 American Chemical Society

in the saturation solubility is observed, especially for nanoparticles with a size less than 100 nm.13 However, drug nanosuspensions often suffer from poor physicochemical stability, with the possibility of the particle size increasing during storage. Agglomeration and crystal growth of a drug adversely affect its dissolution and absorption behavior because they decrease the surface area-to-volume ratio.14 Thus, addition of stabilizers into the system has been widely implemented to obtain nanosuspensions with long-term stability.15−17 Typical stabilizers used are polymers, such as polyvinylpyrrolidone (PVP),6 hypromellose (HPMC),18 and hypromellose acetate succinate,19 the ionic surfactant sodium dodecyl sulfate (SDS),20 and the nonionic surfactants poloxamer21 and Tween 80.22 It is well-known that the anionic surfactant SDS forms a complex with PVP23,24 and HPMC25,26 as a necklacelike structure above a critical aggregation concentration (cac). This unique complex stabilizes drug particles by covering the Received: Revised: Accepted: Published: 2972

March 25, 2015 June 3, 2015 June 24, 2015 June 24, 2015 DOI: 10.1021/acs.molpharmaceut.5b00236 Mol. Pharmaceutics 2015, 12, 2972−2980

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Molecular Pharmaceutics

dodecyl sulfate (SDS; Mw 288.38) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The chemical structures of PBC, HPMC, and SDS are shown in Figure 1. All of the other chemicals used were of reagent grade.

colloidal surface. Although there have been many studies related to the stabilization of drug nanosuspensions using excipients, the agglomeration behavior of nanoparticles in aqueous media is not yet fully understood. Thus, to date, the trial-and-error approach is the only method of preparing nanosuspensions with long-term stability. Therefore, it is expected that elucidating the mechanism of agglomeration on the molecular level will contribute to more effectively designing and formulating stable nanosuspensions. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are generally applied techniques to observe the morphology of nanoparticles. However, neither of these measurements can be used to perform a straightforward evaluation of the properties of nanoparticles in an aqueous environment because the sample should be completely dried before capturing the sample’s image.27,28 Although cryogenic SEM and cryogenic TEM are also employed for observing samples in a suspended state,29,30 the rapid freezing process may negatively affect the molecular state of the samples. Atomic force microscopy (AFM) is a promising approach for observing suspended samples that provides information on the 3D topography of the sample on a nanoscale by scanning over the surface.31 It is possible to directly evaluate the characteristics of nanoparticles in aqueous media because this method is applicable under both ambient and fluidic conditions. Moreover, AFM force−distance curve analysis is a powerful method to probe the mechanical properties of a sample.31,32 Yamamoto et al. have investigated the growth mechanism of the soap-free polymerization of styrene using force−distance curve measurements.33,34 In the pharmaceutical field, the study of drug encapsulation into a liposome using AFM has been reported by Ramachandran et al.35 For in vitro and in vivo applications, the force of nanoparticle−cell membrane interactions is quantitatively evaluated using a surface-modified tip.36 Moribe et al. have examined the physical properties of a nanosuspension from a dry co-ground mixture (GM) using AFM measurements, where the influence of the polymer species and the grinding method on the surface structure of the nanoparticles was investigated.37,38 The difference in the nanoparticle component arising from subtle structural distinctions between phenytoin and its derivatives was also evaluated.39 The purpose of this study is to clarify the agglomeration mechanism of nanoparticles suspended in water during storage. In this study, probucol (PBC), an antihyperlipidemic drug, was used as a poorly water-soluble drug. HPMC and SDS were utilized as a water-soluble polymer and an anionic surfactant, respectively. These three materials were concurrently spraydried, and the ternary spray-dried sample (SPD) was dispersed in water to prepare the nanosuspension. First, the molecular states of the ternary SPD were investigated by powder X-ray diffraction (PXRD) and solid-state 13C NMR measurements. Then, AFM measurements were employed for the direct evaluation of the agglomeration behavior and mechanical properties of the nanoparticles in aqueous solution. Furthermore, the molecular states of the freeze-dried (FD) nanosuspensions were evaluated by PXRD and solid-state 13C NMR measurements to confirm the AFM results.

Figure 1. Molecular structures of (a) probucol (PBC), (b) hypromellose (HPMC), and (c) sodium dodecyl sulfate (SDS).

Methods. Preparation of the Physical Mixture (PM) and the Spray-Dried Sample (SPD). PBC, HPMC, and SDS were physically mixed at a weight ratio of 1:1.75:1.25 in a glass vial using a vortex mixer for 3 min to prepare the ternary physical mixture (PM). The PM was dissolved in a dichloromethane/ methanol = 1:1 (v/v) solution at a concentration of 5% w/v. The solution was spray-dried using an ADL311S spray dryer (Yamato Scientific Co., Ltd. Tokyo, Japan). The inlet temperature of 65 °C, the atomizing pressure of 0.05 MPa, and the feeding rate of 4 g/min were held constant throughout the experiments. Preparation of SPD Suspension. The SPD was dispersed into distilled water with a PBC concentration of 0.5 mg/mL and then sonicated for 3 min to form the nanosuspension. The SPD suspension was stored at 25 °C, and the change in the size of the particles during storage was evaluated. Preparation of Freeze-Dried Sample (FD). The SPD suspensions after storage at 25 °C for 0, 1, and 7 days were freeze-dried using a DRC-1100 freeze dryer (Tokyo Rikakikai Co., Ltd. Tokyo, Japan). Samples that were freeze-dried after storage for 0, 1, and 7 days were abbreviated as FD (freshly prepared), FD (1 day stored), and FD (7 day stored), respectively. The freeze-drying conditions were as follows: prior to freezing, the samples were held at −40 °C for 40 min; pressure reduction, −40 °C for 1.5 h; primary drying, −20 °C for 12 h; and secondary drying, 20 °C for 5 h. Powder X-ray Diffraction (PXRD) Measurements. PXRD measurements were performed on a MiniFlex II diffractometer (Rigaku Co. Tokyo, Japan) with the following experimental conditions: target, Cu; filter, Ni; voltage, 30 kV; current, 15 mA; scanning angle, 3−40°; and scanning speed, 4°/min. Solid-State 13C NMR Measurements. Solid-state 13C NMR measurements were performed on a JNM-ECX400 NMR spectrometer with a magnetic field at 9.39 T (JEOL Resonance Co., Ltd. Tokyo, Japan). The pulse sequence of crosspolarization and the total suppression of spinning sidebands

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EXPERIMENTAL SECTION Materials. Probucol (PBC; Mw 516.84, Form I) was supplied by Daiichi-Sankyo Co., Ltd. (Tokyo, Japan). Hypromellose (HPMC; Type TC-5E, Mw ≈ 12 600) was a gift from the Shin-Etsu Chemical Co. (Tokyo, Japan). Sodium 2973

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Molecular Pharmaceutics were utilized under magic-angle spinning (MAS) conditions. NMR measurement conditions were as follows: contact time, 5 ms; data points, 2048; relaxation delay, 1−2 s; scans, 2002− 30008 times; and spinning rate, 5 kHz. All of the NMR spectra were externally referenced by setting the methyl peak of hexamethylbenzene to 17.3 ppm. The numbers shown at the PBC40 and SDS41 carbons in Figure 1 indicate the peak assignments for the solid-state 13C NMR spectra. Particle Size Distribution Measurement. The volumetric mean particle size (mv) of the suspension was determined using the dynamic light scattering method with a Microtrac UPA (Nikkiso. Co. Tokyo, Japan; measurement range: 0.0008−6.5 μm). Solution-State 1H NMR Measurement. Solution-state 1H NMR measurements were performed using a JNM-ECA500 NMR spectrometer with a magnetic field at 11.74 T (JEOL Resonance Co., Ltd. Tokyo, Japan). NMR spectra were obtained under the following conditions: data points, 32 768; relaxation delay, 5−15 s; scans, 64−256; and spinning rate, 15 Hz. Trimethylsilyl propionate (TSP) was used as an internal reference. The calibration curves were constructed using an HPMC and SDS solution as a standard. The peak area ratio of the respective peaks in the low (2.9−4.2 ppm) and high (0.6− 1.9 ppm) magnetic field was plotted versus that of the TSP peak. All of the calibration curves exhibited good linearity (r2 > 0.995). Negatively Stained Field Emission-Transmission Electron Microscopy (FE-TEM) Measurement. The morphology of the suspended nanoparticles was investigated using field emissiontransmission electron microscopy (FE-TEM), JEM-2100F (JEOL Co., Ltd. Tokyo, Japan), with an accelerating voltage of 120 kV. The TEM grid (Cu200, Nisshin EM Co. Ltd., Japan) was hydrophilized for 40 s using an HDT-400 hydrophilic treatment device (JEOL Co., Ltd. Tokyo, Japan). Subsequently, the SPD suspension was adsorbed on the grid for 1 min. After removing excess solution, 2% phosphotungstic acid (pH 7.4) was adsorbed onto the grid for 40 s to continuously remove the excess stain. The TEM grid was dried overnight in a desiccator under vacuum before the FE-TEM measurement. Atomic Force Microscopy (AFM) Measurement. An AFM apparatus, MFP-3D (Oxford Instruments, Osaka, Japan), was used to evaluate the topography and stiffness of the nanoparticles in an aqueous solution. The cleaved mica surface was modified with (3-aminopropyl)triethoxysilane (APTES) to create a positively charged surface. A positively charged mica surface immobilizes negatively charged drug nanoparticles electrostatically. The sample suspension (ca. 50 μL) was dropped onto the positively charged AP-mica surface and incubated for specific time intervals. The excess particles, which were not immobilized, were washed away by additional water before the AFM measurement was recorded. Topographical images, height images, and force−distance curves of the nanoparticles adsorbed on the mica surface were obtained with a cantilever, TR400PSA (Olympus Co., Ltd. Tokyo, Japan), with a spring constant of 0.08 N/m. The other AFM measurement conditions were as follows: image pixels, 1024 × 128; scanning speed, 0.50 Hz; temperature, 25 °C; and trigger point, 5 nN.

Figure 2. Powder X-ray diffraction (PXRD) patterns of (a) intact PBC (Form I; ■), (b) spray-dried PBC (Form II; ▲), (c) HPMC, (d) SDS (●), (e) spray-dried SDS (⧫), (f) ternary PM, (g) binary SPD of PBC/HPMC, (h) binary SPD of PBC/SDS, (i) binary SPD of HPMC/SDS, (j) ternary SPD, (k) FD (freshly prepared), (l) FD (1 day stored), and (m) FD (7 day stored).

intact PBC (Figure 2a). Gerber et al. have reported that PBC has two polymorphs: Form I (stable) and Form II (metastable).42 In PBC Form II, the C−S−C−S−C chain is extended and the molecule exists as an symmetrical structure. Conversely, this symmetry is lost in PBC Form I, in which the torsion angles around the C1−S1 and C1−S2 bonds significantly deviate from 180°. The diffraction pattern of spray-dried PBC was consistent with the reported pattern for PBC Form II.41 Therefore, metastable PBC Form II was obtained by applying the spray-drying method. Moreover, a polymorph of SDS was verified by spray drying (Figure 2e). A halo pattern was observed for the binary SPD of PBC/HPMC, indicating that PBC was dispersed in the HPMC matrix in an amorphous state (Figure 2g). In contrast, the binary SPD of PBC/SDS showed characteristic diffraction peaks of PBC and SDS crystals (Figure 2h). SDS peaks were still observed in the binary SPD of HPMC/SDS. However, the peak intensity of SDS was significantly decreased (Figure 2i). Although the ternary PM showed a superimposition pattern of each component (Figure 2f), no diffraction peaks derived from the

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RESULTS AND DISCUSSION Molecular State of SPD. Figure 2 shows the powder X-ray diffraction (PXRD) patterns of each sample. Spray-dried PBC (Figure 2b) exhibited a different diffraction pattern from that of 2974

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the interaction mechanism could be different between the PBC/PVP and PBC/HPMC systems. Particle Size of the Freshly Prepared Suspension. The volumetric mean particle size (mv) and the particle size distribution patterns are displayed in Table S1 and Figure S2, respectively. The ternary PM suspension exhibited a white turbid color, and nanoparticles did not form. Moreover, microsized particles were also obtained in the case of the dispersed binary SPD of PBC/HPMC or the binary SPD of PBC/SDS in water (data not shown). However, nanoparticles with a particle size of 245.5 nm were acquired after dispersion of the binary SPD of PBC/HPMC in the SDS solution (Figure S2a). The mv of the freshly prepared ternary SPD suspension was determined to be 25 nm, confirming the formation of small particles (Figure S2b). Hence, it is demonstrated that the coexistence of PBC/HPMC/SDS in solution was essential for the preparation of the nanosuspension. Furthermore, a nanosuspension containing particles with sizes less than 30 nm was prepared only when the three components were simultaneously present during the spray-drying process. Pongpeerapat et al. achieved the preparation of a nanosuspension by employing ternary PBC/PVP/SDS GM.6 The size of the nanoparticles obtained from the ternary GM, especially using low molecular weight PVP K12, was significantly reduced to 16 nm. From these results, a PBC nanosuspension with a very small particle size could be obtained by a top-down approach, such as grinding, and by a bottom-up approach, such as spray drying, with a ternary drug/ polymer/surfactant system. Molecular States of the Freshly Prepared Suspension. The molecular state of the freshly prepared suspension was evaluated by solution-state 1H NMR measurements. Figure 4

PBC crystal were detected in the ternary SPD (Figure 2j). Thus, PBC existed in an amorphous form after spray drying with HPMC and SDS. In contrast, the SDS crystal remained in the ternary SPD, as evidenced by the identification of the diffraction peaks. Figure 3 shows the expanded solid-state 13C NMR spectra (115−160 ppm). The full spectra (0−200 ppm) of the samples,

Figure 3. Solid-state 13C NMR spectra (115−160 ppm) of (a) intact PBC (Form I), (b) spray-dried PBC (Form II), (c) ternary PM, (d) ternary SPD, (e) FD (freshly prepared), (f) FD (1 day stored), and (g) FD (7 day stored).

including intact, binary SPDs, and ternary SPD, are shown in Figure S1. An upfield shift of C4 and a change in some peak shapes were observed for the spectrum of spray-dried PBC (Figure 3b) compared with that of intact PBC (Figure 3a). The spectrum of spray-dried PBC was in good agreement with the reported spectrum of PBC Form II,41 supporting the PXRD results. The ternary PM (Figure 3c) showed a nearly identical spectrum to that of intact PBC, whereas all of the PBC peaks were substantially broadened in the spectrum of SPD (Figure 3d). Geppi et al. reported that signal broadening in the spectrum of a drug in a solid dispersion compared to that for the pure drug was observed because of a wider distribution of isotropic chemical shifts resulting from the higher disorder induced by the occurrence of drug/amorphous polymer interactions.43 Therefore, the peak broadening should indicate the amorphization of PBC in SPD. Pongpeerapat et al. have evaluated the molecular interaction among PBC/PVP/SDS in the ternary GM by solid-state 13C NMR spectroscopy.41 The ternary GM exhibited a new broad NMR peak at approximately 143 ppm, suggesting a change in the chemical environment in PBC due to electrostatic interactions with PVP. However, none of the new peaks derived from the interaction between PBC and HPMC were observed for the spectrum of the PBC/ HPMC/SDS ternary SPD. This result is presumably because

Figure 4. Solution-state 1H NMR spectra of (a) HPMC solution, (b) SDS solution, (c) ternary PM suspension, and (d) ternary SPD suspension at 25 °C.

shows the expanded solution-state 1H NMR spectra in which the peaks of the three components are observed. The full 1H NMR spectra (0−8 ppm) are represented in Figure S3. In the spectrum of the HPMC (Figure 4a) and SDS (Figure 4b) solutions, the respective peaks were clearly detected because they completely dissolved in D2O. However, no PBC peaks were detected in the spectrum of the PBC solution because of its low solubility in water (5 ng/mL at 25 °C).44 The ternary 2975

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Molecular Pharmaceutics PM exhibited a superimposition spectrum derived from HPMC and SDS (Figure 4c). It has been previously reported that the HPMC/SDS necklace-like complex is formed above the cac (3.4−3.6 mM at 25 °C).45 The SDS concentration in the PM suspension was 0.625 mg/mL (ca. 2.2 mM), which was lower than its cac; it was suggested that there were only slight interactions between them. However, in the ternary SPD suspension, all of the SDS peaks were notably broadened, although the HPMC peaks did not change (Figure 4d). This peak broadening could be attributed to the alternating changes of SDS between its unbound and bound states to the surface of the nanoparticles. This exchange was faster than the NMR time scale, causing the NMR signal to appear as a broadened peak, derived from the average of the sharp and very broad peaks. Aiming to further investigate the structure of the particles, quantification of the HPMC and SDS concentrations in the suspension was performed. In the PM suspension, the concentration of HPMC and SDS determined by solutionstate 1H NMR was 0.872 and 0.615 mg/mL, corresponding to 99.6 and 98.4% of the loaded concentrations, respectively (Table 1). This result indicated that most of the stabilizers were Table 1. HPMC and SDS Concentrations of PM and SPD Suspensions Determined by Solution-State 1H NMR Measurementsa PM suspension SPD Suspension

HPMC conc. (mg/mL)

SDS conc. (mg/mL)

0.872 ± 0.004 (99.6%) 0.669 ± 0.004 (76.4%)

0.615 ± 0.002 (98.4%) 0.605 ± 0.007 (96.9%)

n = 3; mean ± SD. Round brackets indicate the ratio of dissolved HPMC and SDS in suspensions against the loaded concentration.

a

Figure 5. Particle size distribution patterns of (a) freshly prepared, (b) 1 day stored, (c) 2 day stored, (d) 4 day stored, and (e) 7 day stored nanosuspensions.

sufficiently mobile, as reflected in the solution-state 1H NMR spectrum. However, in the freshly prepared SPD suspension, the concentration of HPMC decreased to 0.669 mg/mL (76.4%), whereas that of SDS was 0.605 mg/mL (96.9%). It is assumed that approximately one-quarter of HPMC resided in the particle, forming PBC/HPMC mixed nanoparticles in the SPD suspension. Meanwhile, most of the SDS was freely dissolved or was near the surface of the nanoparticles, instead of inside the particle. The SDS localized on the particle’s surface should play an important role in stabilizing the nanoparticles in the suspended state. Additionally, the broadened PBC peak derived from aromatic protons was subtly observed at approximately 7.6 ppm only in the SPD suspension, indicating that the concentration of dissolved PBC was increased. Accurately quantifying the PBC concentration was difficult because of the poor signal-to-noise ratio (S/N) of the obtained peak. However, the approximate concentration was calculated by comparing the peak area of the PBC aromatic carbon with that of TSP. The PBC concentration in the SPD suspension was determined to be approximately 15−25 μg/mL, which was equivalent to 3−5% of the loaded concentration of PBC at 500 μg/mL. Accordingly, the ratio of supersaturated PBC was low compared with the entire PBC in the suspension, although the concentration was drastically increased compared with the solubility at 5 ng/mL. Changes in Particle Size and Mechanical Property of Particles in Nanosuspension during Storage. Figure 5 shows changes in the size distribution of the nanosuspension stored at 25 °C. The particle size of the freshly prepared nanosuspension was 25.0 nm, whereas that of the 1 day stored nanosuspension increased up to 52.5 nm. For the 2 and 4 day

stored nanosuspensions, the particle sizes were 61.5 and 72.4 nm, respectively. Finally, the particle size changed to 93.9 nm after storage for 7 days. Thus, a gradual size increase of the nanoparticles occurred during storage. The morphology of the particles in the nanosuspension was evaluated by FE-TEM measurements (Figure S4). The three FE-TEM images showed spherical particles with a particle size that was generally consistent with the result of the particle size measurement by dynamic light scattering. AFM measurements were subsequently performed to evaluate the agglomeration behavior and mechanical properties of the nanoparticles in detail. Figure 6 shows the AFM topographical and height images of the nanosuspension after being freshly prepared and after storage at 25 °C. In all of the AFM images, spherical particles were observed, coinciding with the result of the FE-TEM measurement. Particle size growth during storage was also confirmed. Therefore, the AFM measurement directly proved the agglomeration process of the nanoparticles under aqueous conditions. Figure 7 shows results of the AFM force−distance curve measurements for 50 particles in each suspension. The slope of the force−distance curves is dependent on the mechanical properties of the sample. Hard materials show a vertical line since the tip hardly indents into the sample, such as for a mica substrate. Meanwhile, for soft materials, the tip completely indents into the samples, and a vertical line is subsequently obtained due to the contact with the hard substrate. Therefore, the force required for full indentation increases with the sample’s stiffness. The freshly prepared nanosuspension exhibited an average height of 15.3 ± 4.4 nm (Table 2). A 2976

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Figure 6. AFM topographical and height images of (a) freshly prepared, (b) 1 day stored, (c) 2 day stored, (d) 4 day stored, and (e) 7 day stored nanosuspensions. Figure 7. AFM force−distance curves of (a) freshly prepared, (b) 1 day stored, (c) 2 day stored, (d) 4 day stored, and (e) 7 day stored nanosuspensions (n = 50).

complete indentation of the tip into the particles within an applied force of 1 nN was detected for all 50 particles. This result indicated that the nanoparticle’s structure was soft compared with the mica substrate. The average height of the 1 day stored nanosuspension was 22.4 ± 6.6 nm. Although complete indentation into particles was also observed for all of the particles, the required force varied by particle. Among them, the majority of the particles completely indented within a force load of 2 nN. The average height of the 2 day stored nanosuspension particles was determined to be 34.0 ± 9.5 nm. Similar to the 1 day stored results, the force−distance curve results differed depending on the particles. The number of particles fully indented within a load force of 1 nN decreased to 6, whereas those requiring 1−3 nN increased to 34. The average height of the 4 day stored nanosuspension particles was calculated to be 43.4 ± 9.6 nm. During this storage period, most of the particles showed a new force−distance curve pattern, indicating that they were partially indented, but the indentation distance was short compared with the particle height. At the end of storage, the average height of the 7 day stored nanosuspension particles was 78.2 ± 12.9 nm. After the 7 day storage, for all 50 particles, nearly no indentation of the tip into the particles was detected, even when applying 5 nN.

Table 2. Number of Particles Classified in Accordance with the Force Required for the Indentation and Average Height of Particles in PBC Nanosuspensionsa force (nN) 5+

height (nm) (mean ± SD)

0

0

15.3 ± 4.4

6

1

0

22.6 ± 6.6

18

9

0

1

34.0 ± 9.5

1

5

4

5

35

43.4 ± 9.6

0

0

0

0

50

78.2 ± 12.9

sample

0−1

1−2

2−3

freshly prepared nanosuspension 1 day stored nanosuspension 2 day stored nanosuspension 4 day stored nanosuspension 7 day stored nanosuspension

50

0

0

0

15

19

9

6

16

0 0

a

3−4 4−5

n = 50.

Figure 8a shows representative AFM force−distance curve patterns of the stored nanoparticles. Using AFM force curve 2977

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to the presence of a small fraction of crystals in the particle, although indentation occurs such that it is almost the same as the particle height. The slope of the AFM force curve shown in Figure 8 is inclined to be steeper on storage, and complete indentation of the tip was observed for particles such as those stored for 1 or 2 days, for which crystallizing is in progress. Therefore, it is suggested that crystallization of nanoparticles in the SPD suspension proceeded via the (B) pattern rather than the (A) pattern. Molecular State of FDs. Freeze-dried samples were prepared to support the AFM results which demonstrated that PBC in the nanoparticles gradually crystallized as the agglomeration behavior occurred. A particle size distribution measurement was conducted to confirm the redispersibility of the FDs before evaluating their molecular state (Figure S5). The particle sizes of FD (freshly prepared), FD (1 day stored), and FD (7 day stored) suspensions were 34.0, 65.6, and 105.4 nm, respectively. These values were nearly equal to the particle size in the nanosuspension (Figure 5). Therefore, the molecular state of PBC in the FD samples was not altered by the freezedrying process. Figure 2k−m shows the PXRD patterns of the FD samples. The ternary SPD showed the only characteristic peaks of an SDS crystal, whereas no diffraction peak of the PBC crystal was detected (Figure 2j). However, FD (freshly prepared) exhibited slight peaks derived from PBC Form II. Moreover, it was obvious that the intensity of the PBC peaks increased from FD (freshly prepared) to FD (7 day stored). The peak intensity in the PXRD pattern is subject to the crystal size and the crystallinity of sample; thus, it was suggested that the observed intensity enhancement of the PBC peaks originated from both factors. Figure 3e−g shows the expanded solid-state 13C NMR spectra (115−160 ppm) of the FD samples. FD (freshly prepared) exhibited a broader peak (123.4 ppm) attributed to amorphous PBC and only slightly exhibited the characteristic peak of PBC Form II crystal (118.5 ppm). Conversely, in the spectrum of FD (1 day stored), the peak intensity of amorphous PBC decreased, whereas that of PBC Form II increased. This trend was more clearly identified in the FD (7 day stored) spectrum. Hence, it was revealed that the molecular state of PBC changed from amorphous to a metastable Form II crystal with storage time.

Figure 8. (a) Representative force−distance curve patterns observed by AFM measurements and (b) proposed changes in the molecular state of PBC in the nanosuspension during storage.

measurements, the force−distance curves were predominantly classified into four patterns, depending on the storage period, as follows: (I) complete indentation of the tip into particles within an applied force of 1 nN, (II) complete indentation of the tip into particles with an applied force of 1−5 nN, (III) partial indentation of the tip into particles with an applied force of 5 nN, and (IV) nearly no indentation of the tip into particles with an applied force of 5 nN. Moreover, the slope of the force curve became steeper with as the force curve patterns changed from (I) to (IV). Broman et al. have evaluated the mechanical property of amorphous and crystalline sorbitol by AFM measurements in the solid state.46 A greater indentation of the tip occurred in the amorphous region than in the crystalline domain, indicating the softer nature of the amorphous materials. Therefore, amorphous PBC particles could be formed in the freshly prepared nanosuspension (Figure 8b). The glass transition temperature (Tg) of amorphous probucol was approximately 26 °C,47 denoting the difficulty in maintaining the amorphous state as PBC alone, especially in the suspension. The coexistence of PBC and HPMC in the particles was found from the solution-state 1H NMR data in Table 1. Thus, it was surmised that the amorphous state of PBC was temporarily retained by the crystallization inhibition efficiency of HPMC47 within the particles. Subsequently, crystallization of PBC gently proceeded with time and was complete after storage for 7 days. To the best of our knowledge, this is the first report to directly investigate the crystallization behavior of a drug in a nanoparticle under aqueous conditions. Here, we proposed two patterns of drug crystallization as follows: (A) outward growth of a single crystal which is formed in the bulk and (B) progression of crystallization and growth simultaneously at various sites in the bulk. In the case of (A), nanoparticles contain separate two domains: an almost completely crystallized part and an amorphous part. Thus, the particles could have their remaining crystalline area located under the tip, where it is too hard to indent them. Conversely, these domains in the nanoparticles could be mixed in the (B) pattern. Consequently, the loading force required increases due

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CONCLUSIONS PXRD and solid-state 13C NMR measurements revealed that PBC existed in an amorphous state in ternary PBC/HPMC/ SDS SPD. A nanosuspension with a substantially smaller particle size of 25 nm was prepared by dispersing ternary SPD into water. For the freshly prepared nanosuspension, a decrease of the HPMC concentration was detected by solution-state 1H NMR measurements. This result indicated the presence of HPMC in the nanoparticles, which contributed to the stabilization of amorphous PBC. Conversely, the calculated concentration of SDS in the freshly prepared nanosuspension was nearly the same as the loaded concentration, suggesting that SDS was freely dissolved or was at the particle’s surface. Meanwhile, a gradual increase in the size of the nanoparticles was observed, with the particle size increasing to 93.9 nm after storage at 25 °C for 7 days. The morphology of the nanoparticles and their agglomeration behavior in water were directly observed from AFM topographical and height images. Moreover, the AFM force−distance curves changed, depending on the storage period, as follows: (I) complete indentation of 2978

DOI: 10.1021/acs.molpharmaceut.5b00236 Mol. Pharmaceutics 2015, 12, 2972−2980

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Molecular Pharmaceutics

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the tip into particles within an applied force of 1 nN, (II) complete indentation of the tip into particles with an applied force of 1−5 nN, (III) partial indentation of the tip into particles with an applied force of 5 nN, and (IV) nearly no indentation of the tip into particles with an applied force of 5 nN. An increase in the force curve’s slope was also observed from (I) to (IV). These results indicated the stiffness increase of the nanoparticles, revealing the gradual transformation of PBC from an amorphous to a crystalline state. Solid-state 13C NMR measurements of FD samples further demonstrated the generation of metastable PBC Form II crystals during storage. Currently, intense focus is being given to nanosuspensions as a pharmaceutical formulation; however, there are few studies investigating the molecular states of nanoparticles in the suspended state. We previously reported the direct evaluation of nanosuspensions at the molecular level by suspended-state NMR.29 However, this methodology requires some constraints, such as high sample concentration and long measurement time. Moreover, only the averaged data from the total system is obtained, causing difficulty in particle differentiation. AFM measurements provide detailed information on every particle by evaluating the mechanical properties of each sample. Finally, we expect that clarifying the agglomeration mechanism utilizing a combination of AFM and other techniques will open doors to the straightforward preparation of stable nanosuspensions.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 shows the full solid-state 13C NMR spectra (0−200 ppm) of samples, including intact, binary SPDs, and ternary SPD. Table S1 and Figure S2 represent the mv and particle size distribution patterns of the suspension of binary PBC/HPMC SPD + SDS and ternary SPD. Figure S3 exhibits the full solution-state 1H NMR spectra (0−8 ppm). Figure S4 shows the FE-TEM images of the respective nanosuspensions. Figure S5 displays particle size distribution patterns of FD suspensions. These materials are available free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.molpharmaceut.5b00236.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-43-226-2865; Fax: +81-43-226-2867; E-mail: [email protected]. Author Contributions †

K.E. and K.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by a Grant-in Aid from the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) of Japan and from Hosokawa Powder Technology Foundation. We would like to thank the Shin-Etsu Chemical Co. (Tokyo, Japan) for the gift of HPMC.

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REFERENCES

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DOI: 10.1021/acs.molpharmaceut.5b00236 Mol. Pharmaceutics 2015, 12, 2972−2980

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DOI: 10.1021/acs.molpharmaceut.5b00236 Mol. Pharmaceutics 2015, 12, 2972−2980