Morphological and physicochemical evaluation of two distinct

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Morphological and physicochemical evaluation of two distinct glibenclamide/ hypromellose amorphous nanoparticles prepared by the antisolvent method Hazuki Yonashiro, Kenjirou Higashi, Chikako Morikawa, Keisuke Ueda, Tsutomu Itoh, Masataka Ito, Hyuma Masu, Shuji Noguchi, and Kunikazu Moribe Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01122 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Molecular Pharmaceutics

Morphological and physicochemical evaluation of two distinct glibenclamide/hypromellose amorphous nanoparticles prepared by the antisolvent method Hazuki Yonashiro†, Kenjirou Higashi∗†, Chikako Morikawa†, Keisuke Ueda†, Tsutomu Itoh‡, Masataka Ito§, Hyuma Masu‡, Shuji Noguchi§, and Kunikazu Moribe† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku,

Chiba 260-8675, Japan; ‡

Center for Analytical Instrumentation, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba

263-8522, Japan; §

Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba

274-8510, Japan

∗

Corresponding Author: Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan; Tel.: +81-43-226-2866; Fax: +81-43-226-2867; E-mail: [email protected]

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

The morphology and stability of amorphous nanoparticles of glibenclamide (GLB) prepared by the antisolvent method using different methods of adding hypromellose (HPMC) were evaluated. Nano-A was prepared by the injection of a dimethyl sulfoxide (DMSO) solution of GLB into the HPMC solution, whereas nano-B was obtained by the injection of a DMSO solution of GLB and HPMC into water. Cryogenic transmission electron microscopy, field-emission scanning electron microscopy, and field-emission transmission electron microscopy including energy dispersive X-ray spectrometry revealed that the particles of the nano-A and nano-B samples are hollow spheres and non-spherical nanoparticles, respectively. Powder X-ray diffraction and solid-state NMR measurements showed that GLB is present in an amorphous state in both nano-A and nano-B. The weight ratios of HPMC in the GLB/HPMC nanoparticles were 11% and 16% for nano-A and nano-B respectively, as determined by solution-state NMR. The glass transition temperatures (Tg) of nano-A and nano-B evaluated using differential scanning calorimetry were lower by ca. 10 °C compared to that of amorphous GLB, presumably because of a Tg confinement effect and the surface coverage and mixing of HPMC, as suggested by the


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inverse gas chromatography experiment. GLB crystallization during storage was suppressed more strongly in nano-B than nano-A owing to the higher amount of HPMC and the higher miscibility between GLB and HPMC. It is suggested that the diffusion rate of the solvent during nanoprecipitation determined the nanoparticle properties. In nano-A, the precipitation of GLB first occurred at the outer interface because of the rapid diffusion of the solvent. Thus, hollow spherical particles with HPMC preferentially located near the surface were formed. On the other hand, the diffusion of the solvent in nano-B was suppressed because of the presence of HPMC, yielding small non-spherical nanoparticles with a high miscibility of GLB and HPMC.

KEYWORDS

Poorly water-soluble drug, amorphous nanoparticle, nanoprecipitation, hollow nanoparticle, drug-polymer miscibility, solvent diffusion rate, Tg confinement effect, surface energy

Introduction In recent years, more than 70% of candidate drugs have been found to show poor water solubility.1 For this reason, various pharmaceutical methods for improving solubility have 3 ACS Paragon Plus Environment


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been developed. Among them, the preparation of drug amorphous nanoparticles has attracted attention.2 Such nanoparticles dissolve rapidly because of the increase in the specific surface area accompanying nanosizing.3,4 Furthermore, because the drug exists in an amorphous state, high supersaturation is achieved on dispersion in water.5 It has been reported that drug amorphous nanoparticles improve drug absorption remarkably compared to drug nanocrystals and drug amorphous microparticles for oral and pulmonary administration.6–8 The preparation of nanoparticles can be classified into two categories based on top-down or bottom-up methods. In the top-down method, the nanosizing of a drug crystal is carried out by a mechanical force using wet-milling9 or high-pressure homogenization.10 Because of the convenience of scale-up, all formulations including the nanocrystalline drugs currently on the market are prepared by the top-down method.11 On the other hand, in the bottom-up method, nanoparticles are precipitated from a solvent in which the drugs are dissolved.12 Although there are some reports of drug amorphous nanoparticles prepared by the top-down method,13,14 these nanoparticles are mainly prepared by the bottom-up method because, in the top-down method, it is difficult to achieve both nanosizing and amorphization of crystalline drugs using mechanical energy. Furthermore, the drugs 4 ACS Paragon Plus Environment

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recrystallize rapidly when crystals are present even in a small amount in the suspension. The bottom-up methods include the use of antisolvents,15–18 reactive precipitation,19,20 solvent

evaporation,21

pH

shift,22

sonoprecipitation23

and

drug-polyelectrolyte

complexation.5,24 Among them, the antisolvent method is simple, low cost, and rapid; thus, most studies reported so far have used this method. Without a stabilizer, it is difficult to maintain the drug in an amorphous state, and the control of particle size is challenging because of rapid crystallization and particle growth. Therefore, stabilizers such as water-soluble polymers25 and surfactants8 are commonly used to maintain the amorphous state of the drug and enhance its water dispersibility. Some reports have focused on the preparation, dissolution, and bioavailability of drug amorphous nanoparticles. On the other hand, the number of reports that focus on the mechanism of their formation or stabilization are limited. Lindfors et al. reported a pioneering series of studies concerning amorphous nanoparticles.26–28 They found that Ostwald ripening results in the aggregation of amorphous nanoparticles in water.26 The homogeneous mixing of drugs and excipients in amorphous nanoparticles, as well as the excipients surrounding the nanoparticle interface, play an important role in the inhibition of Ostwald ripening. They developed a technique to measure drug solubility in an amorphous 5 ACS Paragon Plus Environment


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nanosuspension and found that the excipient ratio in the amorphous nanoparticles strongly influences the drug solubility in water.27 In addition, other authors have evaluated the drug distribution in nanoparticles by fluorescence measurements29 as well as the structure at the nanoparticle surface by X-ray photoelectron spectroscopy.30 We have recently observed the distribution of excipients between the bulk water phase and drug amorphous nanoparticles by solution-state NMR spectroscopy.31 As shown in these reports, the miscibility of the drugs and excipients inside the particles as well as localization of excipient on the surface are important for stabilizing drug amorphous nanoparticles in water. The purpose of this research is to clarify the formation and stabilization mechanism of drug amorphous nanoparticles. Glibenclamide (GLB), an antidiabetic drug, shows a low water-solubility but high permeability, and is categorized as class II based on the biopharmaceutical classification system guided by FDA. Thus, the improvement of water-solubility is a critical issue to enhance its bioavailability. Recently, Yu et al. reported the preparation of GLB amorphous nanoparticles by a high-gravity technique.32 The nanoparticles showed rapid GLB dissolution, demonstrating their potential for pharmaceutical application. However, the detailed structure and molecular states were not investigated. In this study, we prepared amorphous nanoparticles of GLB by an antisolvent 6 ACS Paragon Plus Environment

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method using hypromellose (HPMC) as a stabilizer for the dispersion of nanoparticles in water. Hypromellose was added in two different fashions during nanoparticle formation. Cryogenic transmission electron microscopy (cryo-TEM) measurements were used for morphological observation of the nanoparticles in suspension. Furthermore, the freeze-dried nanoparticles were observed by field-emission scanning electron microscopy (FE-SEM) and field-emission transmission electron microscopy (FE-TEM), including scanning transmission electron microscopy-energy dispersive X-ray spectrometry (STEM-EDX). Solid-state and solution-state NMR spectroscopies, powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), N2 adsorption, and inverse gas chromatography (IGC) measurements were used to evaluate the structure and molecular states of the freeze-dried nanoparticles in detail. Finally, the formation and stabilization mechanism of the obtained drug amorphous nanoparticles are discussed based on their morphology, structure, and molecular states.

MATERIALS



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GLB was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). HPMC (type TC-5E, Mw ≈ 12,600) was kindly gifted by Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). All other materials and solvents were of reagent grade. The chemical structures and peak assignments for the 13C NMR spectra of GLB and HPMC are shown in Figure 1.

Figure 1. Chemical structures of (a) glibenclamide (GLB) and (b) hypromellose (HPMC). The carbon numbering of GLB is for the purpose of peak assignment on the NMR spectra.

METHODS 8 ACS Paragon Plus Environment

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Preparation of nano-A and nano-B suspensions Nano-A and nano-B suspensions were prepared by two antisolvent methods. In the preparation method A, 3 mL of a dimethyl sulfoxide (DMSO) solution of GLB (20 mg/mL) was injected into 60 mL of a HPMC solution (0.5 mg/mL) at 2 mL/min while stirring at 20-25 °C to obtain the nano-A suspension. The stirring rate was controlled at 1,800 rpm. In the preparation method B, 3 mL of a DMSO solution of both GLB (20 mg/mL) and HPMC (10 mg/mL) was injected into 60 mL of distilled water under the same conditions to obtain the nano-B suspension. A 100 mL beaker, stirrer (SW-10, Nissinrika, Japan), and stirrer tip 8 mm in diameter and 30 mm in length were used in all the preparations. A syringe pump (YSP-101, YMC Co., Ltd., Japan) and injection needle (25 G 5/8; 0.50 × 16 mm, Terumo, Japan) were used to inject the DMSO solutions.

Freeze-drying of nano-A and nano-B suspensions Nano-A and nano-B were prepared by freeze-drying the corresponding suspensions. The nano-A and nano-B suspensions were centrifuged (150,000 × g) for 40 min, and the supernatant was removed. The freeze-drying of the suspensions was carried out in a dry chamber (DRC-1100/freeze-dryer FDU-2100, EYELA, Japan). Freezing was conducted in 9 ACS Paragon Plus Environment


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a shelf precooled at −40 °C (shelf inlet temperature) for 2 h. After pressure reduction for 2 h, primary and secondary drying was performed under vacuum at −20 °C for 5 h and 20 °C for 5 h, respectively. The residual amount of DMSO was determined after dissolving nano-A and nano-B in d7-dimethylformamide by solution-state NMR spectroscopy. The calculated ratios of DMSO to GLB (v/w%) in nano-A and nano-B were negligibly small, 1.0% and 1.1%, respectively.

Preparation of amorphous GLB Amorphous GLB with a spherical shape and size around several µm (Figure S1) was prepared by spray-drying for comparison with nano-A and nano-B. GLB was dissolved in a dichloromethane/methanol (1:1, v/v) solution at a concentration of 3% (w/v) and fed into a spray dryer (ADL311S; Yamato Scientific Company, Ltd., Tokyo, Japan) at 4 g/min to prepare the amorphous GLB. The solution was spray-dried at an inlet temperature of 90 °C and an atomizing pressure of 0.05 MPa.

Particle size distribution


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The particle sizes of the nano-A and nano-B suspensions were determined using the dynamic light scattering method with a UPA-UT 151 (Nikkiso Co., Ltd., Tokyo, Japan). The measurement conditions are as follows: measurement time, 60 s; repeat count, 3; and temperature, 25 °C.

Cryo-TEM Cryo-TEM measurements of the nano-A and nano-B suspensions were carried out using a JEM-2100F (JEOL Co., Ltd., Japan) at an applied voltage of 120 kV. A collodion film-supported grid Cu200 (Nisshin EM Co. Ltd., Japan) was exposed to a hydrophilic treatment for 1 min, and 2 µL of the suspension were dropped onto the grid. After the removal of excess solution with a filter paper, the grid was rapidly frozen in liquid ethane at about −170 °C. Cryo-TEM measurement was carried out below −170 °C using a Gatan 626 cryo-holder (Gatan, Inc., CA, USA) in liquid nitrogen.

FE-SEM FE-SEM measurements were performed using a JSM-6335F (JEOL Co., Ltd., Japan) at an applied voltage of 5 kV. The sample was fixed on a carbon tape and coated with osmium 11 ACS Paragon Plus Environment


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using a Neoc-ST plasma coater (Meiwaforsis, Japan). The thickness of the osmium coating was kept below 2.5 nm.

FE-TEM FE-TEM measurements were carried out using JEM-2100F (JEOL Co., Ltd., Japan) with an applied voltage of 120 kV. A collodion film-supported grid Cu200 (Nisshin EM Co. Ltd., Japan) was exposed to hydrophilic treatment for 1 min. A small amount of each sample was sprinkled over the grid without removing excess powder. STEM-EDX measurements using a JED-2300 (JEOL Co., Ltd., Japan) were carried out below −170°C, which was achieved using liquid nitrogen and a Gatan 626 cryo-holder (Gatan, Inc., USA), to suppress electron beam damage during the measurement. The diameters (Heywood diameter) of the nanoparticles were analyzed using Image J (ver. 1.41), an open source Java program (n = 500).

Solid-state 13C NMR spectroscopy The solid-state NMR measurements were conducted using a JNM-ECX-400 NMR (9.4 T; JEOL Co., Ltd., Japan) at room temperature. The

13

C NMR spectra were acquired using 12

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cross polarization (CP) with magic-angle spinning (MAS) at 15 kHz. The relaxation delay was set to 3.2–20.0 s based on the 1H-spin-lattice time (T1) of each sample, which was pre-determined using the inversion recovery method. The other conditions were as follows. Decoupling method: two-pulse phase-modulated (TPPM) method, 1H 90º pulse width: 2.95 µs, contact time: 3 ms, and the number of data points: 2048. All the spectra were externally referenced by setting the methane peak of hexamethylbenzene at 17.3 ppm.

PXRD PXRD measurements were performed on a D8 ADVANCE (Bruker AXS, Germany) under the following conditions. Target: Cu, filter: Ni, voltage: 40 kV, current: 40 mA, step size: 0.02º, scanning angle: 5–30º, and time per step: 0.75 s.

Solution-state 1H NMR spectroscopy The freeze-dried samples were dissolved in DMSO-d6 and used as solution-state 1H NMR samples. The solution-state 1H NMR measurements were carried out using a JNM-ECA 500 (11.7 T; JEOL Co., Ltd., Japan) at a spinning rate of 15 Hz at 25 °C. Tetramethylsilane (TMS; 0.0 ppm) was used as an internal standard. A quantitative analysis of the dissolved 13 ACS Paragon Plus Environment


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GLB was performed by comparing the peak area of the GLB methoxy group with that of the TMS methyl group in the 1H NMR spectra. The calibration curve was prepared based on the ratio of the peak area of the GLB methoxy group (y-axis) to that of the TMS methyl group of 1 using GLB solutions (1.33–5.83 mg/mL, x-axis) as the standards. The calibration curve showed good linearity (y = 8.544 x, r2 = 0.992).

DSC The DSC measurements were performed using a DSC 6200 (Seiko Instrument Inc., Japan). For the measurements, 1–5 mg of the powder sample was placed in a crimped aluminum pan with a pinhole. Dry nitrogen was used as the inert gas at a flow rate of 50 mL/min, and the measurements were carried out at 30–200 °C at a heating rate of 10 °C/min.

N2 adsorption The specific surface area of the nanoparticles was calculated by the Brunauer–Emmett– Teller (BET) method using a Macsorb model HM-1201 (Mountech Co., Ltd., Japan) at −196 °C with nitrogen as the adsorption gas.


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IGC Experiments were performed using an iGC-SEA (Surface Measurement Systems, Ltd., UK) by modifying the previously reported methodology.33,34 Samples of mass 100–200 mg were packed into a silanized glass column (Surface Measurement Systems, Ltd., UK) of 6 mm o.d., 4 mm i.d., and 300 mm length. Tapping was carried out for 10 min. Both ends of the column were loosely stoppered with silanized glass wool. The conditioning of the column packed with the sample powder was carried out at 30 °C and 0% relative humidity (RH), and the experiment was performed under these conditions. Methane was used as the inert reference. n-Decane, n-nonane, n-octane, n-heptane, and n-hexane were used to determine the alkane line, whereas chloroform and ethyl acetate were employed as polar probes. The gas flow rate used was 10 mL/min, and helium was used as the carrier gas. The powder surface energy can be calculated from the retention time of the nonpolar and polar probes.

Storage stability tests The amorphous stability of GLB in the freeze-dried samples was evaluated after storage at 25 °C and 60% RH for 4 weeks using aqueous solutions saturated with sodium nitrite. PXRD and TEM measurements were performed at regular intervals. 15 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION Morphologies of the nanoparticles in water and after freeze-drying Figure 2 shows the particle size distribution patterns of the nano-A and nano-B suspensions, as determined by dynamic light scattering. The mean volume diameters (MV) of the freshly prepared nano-A and nano-B suspensions were about 115 and 105 nm, respectively. Both the suspensions showed unimodal size distribution patterns with a polydispersity index (PDI) of around 0.10 (Figures 2a and 2c). The zeta potentials of nano-A and nano-B at -24.5 and -22.7 mV respectively were almost similar to each other (Table S1). In addition, after storage at 4 °C for 24 h, both the suspensions revealed particle size distributions and zeta potentials similar to those of the freshly prepared suspensions (Figures 2b and 2d, and Table S1).


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Figure 2. Particle size distribution patterns of (a, b) nano-A and (c, d) nano-B suspensions. The images a, c and b, d show the patterns of freshly prepared and 24-h-stored samples, respectively. The mean volume diameter (MV ± S.D. n=3) and polydispersity index (PDI) are shown in the figure. The morphologies of the nanoparticles in the freshly prepared suspensions were evaluated by cryo-TEM (Figures 3a and 3c). The shape and size of the particles in the nano-A and nano-B suspensions are different. In the nano-A suspension, spherical particles were observed. The contrast at the particle center is low, suggesting that this center is less dense or hollow. On the other hand, non-spherical particles of uniform contrast were observed in the nano-B suspension. The particle size of the nano-B suspension was slightly smaller than 17 ACS Paragon Plus Environment


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that of the nano-A suspension. Even after static storage at 4 ºC for 24 h, the shape and size of the particles remained similar to those of the freshly prepared suspensions (Figures 3b and 3d). The nanoparticle morphologies in both the suspensions, which differed depending on the method of HPMC addition, were stable on storage.

Figure 3. Cryo-TEM images of (a, b) nano-A and (c, d) nano-B suspensions. The images a, c and b, d represent freshly prepared and 24-h-stored samples, respectively. Each bar represents 200 nm. The morphologies of nano-A and nano-B obtained by centrifugation and freeze-drying of the corresponding nanosuspensions were evaluated by FE-SEM (Figures 4a and 4b) and


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FE-TEM (Figures 4c and 4d). Aggregates of primary particles about 100 nm in diameter were observed in the FE-SEM images of both the samples. The surfaces of the nanoparticles were smooth, and the particle size and shape of each primary particle observed by FE-TEM are similar to those shown in the cryo-TEM images (Figure 3). Hence, for nano-A, spherical and rather large particles with a low contrast at the particle center were observed, whereas non-spherical and rather small particles with a uniform contrast were observed for nano-B. Thus, the particle morphologies were maintained even after the centrifugation and freeze-drying processes. The particle size distributions of the amorphous nanoparticles were evaluated for 500 particles in the FE-TEM images (Figure S2). The calculated mean number diameters (MN) of nano-A and nano-B were about 100 and 90 nm respectively.



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Figure 4. FE-SEM and FE-TEM images of (a, c) nano-A, and (b, d) nano-B, respectively. Each bar represents 200 nm. STEM-EDX measurements were performed on different nanoparticles (Figure 5). Figures 5c and 5d show the Cl distributions, obtained by EDX analysis, of nano-A and nano-B, respectively. The spectral widths of the Cl distribution coincide with the widths of the nano-A and nano-B particles. Cl is present only in GLB, indicating that GLB was distributed throughout the particles. Furthermore, in nano-A, the contrast of the STEM image at the particle center and the intensity of the Cl spectra concurrently decreased, which strongly indicated a hollow structure.


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Figure 5. STEM images and EDX line scans (Cl) of (a, c) nano-A and (b, d) nano-B, respectively. Each bar represents 100 nm. The crystallinity of the GLB in nanoparticles was evaluated by PXRD measurements (Figure 6 left). In the crystalline GLB (Figure 6a), characteristic peaks were found at 2θ values of 11.8, 16.3, and 21.1°. The amorphous GLB prepared by spray-drying showed a halo pattern (Figure 6c). In the PXRD patterns of both nano-A and nano-B, the diffraction peaks of crystalline GLB completely disappeared and showed a halo pattern (Figures 6d and 6e). Therefore, GLB in the nanoparticles could exist in the amorphous state. The molecular states of GLB and HPMC in the nanoparticles was evaluated by solid-state NMR spectroscopy. Figures 6 (right) and S3 show the enlarged (65–175 ppm) and full solid-state 13

C NMR spectra, respectively. Peak assignments are based on a previous report.35 The 21 ACS Paragon Plus Environment


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peaks corresponding to the GLB in the nano-A and nano-B samples were broad and similar to those of the amorphous GLB prepared by spray-drying. In addition, the C7 peak of the GLB carbonyl group shifted up-field for both the nano-A (164 ppm) and nano-B (164 ppm) samples compared to that of crystalline GLB (167 ppm). This change in chemical shift is considered to be due to the amorphization of GLB accompanied by amide-to-imide tautomerism.36,37 In addition to the GLB peaks, HPMC peaks were observed at 70–110 ppm in the spectra of both nano-A and nano-B (enlarged spectra in the insets). Centrifugation at 150,000 × g was performed before freeze-drying to obtain nano-A and nano-B, so the detected HPMC was not just weakly adsorbed on the surface of the nanoparticles. In the antisolvent method, nanoparticles are formed by the precipitation of GLB from the solvent. Hence, HPMC dissolved in aqueous (nano-A) or organic solvents (nano-B) should be incorporated into the nanoparticles when GLB was precipitated as amorphous nanoparticles. This result is consistent with the previous report by Cheow et al., who showed that HPMC is contained in amorphous nanoparticles prepared by the pH-shift precipitation method using itraconazole and HPMC.38


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Figure 6. PXRD patterns (left) and solid-state 13C CP/MAS NMR spectra in the range of 70–170 ppm (right) of (a) crystalline GLB, (b) HPMC, (c) amorphous GLB, (d) nano-A, and (e) nano-B. The insets show the enlarged HPMC peaks. The composition of nano-A and nano-B was determined by solution-state NMR spectroscopy by dissolving the samples in DMSO-d6.39 Figures 7 and S4 show the enlarged (3.40–3.60 ppm) and full solution-state 1H NMR spectra, respectively. Both GLB and HPMC peaks were observed in the nano-A and nano-B spectra (Figures 7c and 7d). The relative peak area of HPMC with respect to the peak of the GLB methoxy group was larger 23 ACS Paragon Plus Environment


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in nano-B compared to that in nano-A. This suggests that nano-B contained a larger amount of HPMC. The amount of dissolved GLB was determined by comparing the peak area of the GLB methoxy group with that of the methyl group of an internal standard (TMS). The weight ratio of GLB/HPMC in the nanoparticles was calculated based on the determined amount of GLB and the powder weight dissolved in DMSO-d6 (Table 1). Nano-A and nano-B had GLB/HPMC weight ratios of 89.0:11.0 and 84.3:15.7, respectively, confirming that the nanoparticles of nano-B contained a greater quantity of HPMC compared to those of nano-A.


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Figure 7. Solution-state 1H NMR spectra in the 3.40–3.60 ppm range for (a) GLB, (b) HPMC, (c) nano-A, and (d) nano-B. All the samples were dissolved in DMSO-d6.

Table 1. HPMC weight ratio in GLB/HPMC nanoparticles as calculated from solution-state 1

H NMR spectra (n = 5, ± S.D.). HPMC weight ratio (%) Nano-A

11.0 ± 2.4*

Nano-B

15.7 ± 3.3*

*p < 0.05 vs each sample



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In the preparation of the nano-A suspension, GLB and HPMC were separately dissolved in DMSO and an aqueous solution, respectively. Therefore, only HPMC near the interface of the DMSO droplet particle and water would be solidified along with GLB. On the other hand, during the preparation of the nano-B suspension, both GLB and HPMC were dissolved in DMSO. Quantitative analysis indicated that 1.0 mg/mL of GLB and 0.19 mg/mL of HPMC were contained in nano-B, and 0.31 mg/mL of HPMC diffused into the aqueous phase accompanying the diffusion of DMSO. Taking the dose concentration of HPMC of 0.50 mg/mL into account, more than 60% of HPMC diffused into the aqueous phase during nanoparticle formation. However, a larger amount of HPMC was still present in nano-B than nano-A. The thermal behavior of nano-A and nano-B were evaluated by DSC measurements. A peak corresponding to melting and a baseline shift due to decomposition after melting were observed at 170 °C in the DSC curve of crystalline GLB (Figure 8a)36,40. In amorphous GLB, a glass transition peak at 73 °C, a crystallization peak near 130 °C, and a melting peak of crystalline GLB around 170 °C and a base line shift above ca. 190 °C (decomposition of GLB) were observed (Figure 8c). In nano-A, a glass transition peak at 63 °C, a crystallization peak near 130 °C, and a melting peak of GLB crystal around 170 °C 26 ACS Paragon Plus Environment

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and a base line shift above ca. 190 °C (decomposition of GLB) were observed (Figure 8d). On the other hand, in nano-B, although a glass transition peak around 63 ºC and a base line shift above ca. 190 °C (decomposition of GLB) was observed, the melting and crystallization peaks were hardly observed (Figure 8e).

Figure 8. DSC curves of (a) crystalline GLB, (b) HPMC, (c) amorphous GLB, (d) nano-A, and (e) nano-B. The arrows in the figure show the glass transition temperature (Tg). The insets show the enlarged curves at the arrow marks. Table 2 summarizes the glass transition temperature (Tg) and the crystallization and melting enthalpies (∆Hc, ∆Hm) of each sample (n = 3). The Tg values of nano-A (62.5 °C) 27 ACS Paragon Plus Environment


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and nano-B (63.0 °C) were lower by ca. 10 °C compared to that of amorphous GLB (72.8 °C). The Tg confinement effect, whereby Tg deviates from the bulk value upon particle size reduction to the nanoscale, has been previously reported in polymer chemistry studies.41 The Tg of nanoconfined polymers can shift by over 50 °C compared to the Tg of the bulk depending on their properties.42 The detailed mechanism of the Tg confinement effect is still a subject of debate, although interfacial effects have been suggested to play an important role,43 and the increased free surface increases the local segmental dynamics,44 which results in a reduction in Tg. The specific surface areas of amorphous GLB, nano-A, and nano-B were determined by N2 adsorption measurements using the BET method (Table S2). The specific surface areas of nano-A (29.3 m2/g) and nano-B (35.0 m2/g) were significantly increased compared to that of amorphous GLB (1.8 m2/g). Interfacial effects increase with increasing interfacial surface-to-volume ratio. Hence, the decline of Tg in both nano-A and nano-B could be due to the increase in the specific surface area accompanying the nanosizing. On the other hand, it is also known that surface coating43 and homogeneous mixing with stabilizers can significantly influence the Tg.45 The Tg confinement effect is suppressed by surface coating with rigid materials, whereas the Tg is enhanced by homogeneous mixing with a high Tg material. We suggest that HPMC, which has a 28 ACS Paragon Plus Environment

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relatively high Tg of around 140 ºC (Figure 8b),46 could be present on the surface and also in the inner core of the nanoparticles. The Tg reduction in nano-A and nano-B by ca. 10 °C relative to amorphous GLB could be a result of the Tg confinement effect on nanosizing and also the surface coating and mixing in the inner core with HPMC. The HPMC in the nanoparticles should not only stabilize the nanoparticle dispersion in the suspension because of steric repulsion but also stabilize the amorphous GLB in the solid owing to the suppression of the depression in Tg. Comparing nano-A and nano-B, the Tg values were almost the same despite their different particle sizes (Figure 4). The amount of HPMC in nano-A was 11.0%, whereas it was significantly higher (15.7%) in nano-B, as determined by 1H NMR measurements (Figure 7). It has been suggested that the similar Tg of the nanoparticles43 could result from a balance of the effects of particle size and amount of HPMC. In both nano-A and nano-B, ∆Hc and ∆Hm decreased compared to that of pure amorphous GLB. In addition, the ∆Hc and ∆Hm in nano-B were significantly lower than those in nano-A, indicating that GLB crystallization in nano-B was further inhibited. Nano-A and nano-B were thermally annealed above the Tg, and their nanostructures were not retained because of coalescence. Thus, the difference in the crystallization behaviors of



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amorphous GLB between the samples upon heating could be simply explained by the amount of HPMC and the miscibility of GLB and HPMC.47,48

Table 2. Glass transition temperature and the crystallization and melting enthalpies of GLB (∆Hc, ∆Hm) as determined from the DSC curves (n = 3, mean ± S.D.). The ∆Hc and ∆Hm of nano-A and nano-B are equivalent considering the GLB weight ratio determined using solution-state 1H NMR spectra. Tg [ºC]

∆Hc [mJ/mg] ∆Hm [mJ/mg]

Crystalline GLB

―

―

94.2 ± 2.3

Amorphous GLB

72.8 ± 0.5

71.9 ± 0.9

77.8 ± 4.9

Nano-A

62.5 ± 2.5

21.3 ± 12.3

27.1 ± 17.9

Nano-B

63.0 ± 2.5

0.5 ± 0.9

1.8 ± 2.1

The storage stability of amorphous GLB in nano-A and nano-B was evaluated under humid conditions at 25 °C, 60% RH (Figure 9). In nano-A, crystallization of GLB was observed after 7 days, and it progressed further for 4 weeks (Figure 9a). As shown in the TEM image of nano-A after four-week storage, recrystallized GLB with a needle-like shape can be seen, as well as the nano-A particles that have maintained their nanostructures


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(Figure 9c). On the other hand, in nano-B, the halo PXRD pattern was maintained even after 4 weeks (Figure 9b). The TEM images confirmed that the morphology of nano-B was retained even after storage (Figure 9d). These PXRD and TEM results indicate that the storage stability of amorphous GLB and the nanostructure was higher in nano-B than nano-A. Drug crystallization is affected by various factors such as Tg, the amount of stabilizer, miscibility, and the surface state.49 In particular, for nanoparticles, the surface state can strongly affect the drug crystallization because of the high specific surface area. Therefore, we used a precise surface characterization technique IGC to investigate the surface state of each nanoparticle (Table 3). The total surface energies (γs) and the dispersive (γsD) and specific components (γsP) of both nanoparticle samples were higher than those of amorphous GLB because of both size reduction and surface coverage of HPMC.50 The γ, γsD, and γsP of nano-A and nano-B are in good agreement despite their different particle sizes. The HPMC surface coating possibly reduces the effects of the differences in particle size between nano-A and nano-B on the surface energy. Therefore, the higher stability of amorphous GLB in nano-B than nano-A after storage could be due to the higher amount of HPMC and the miscibility of GLB and HPMC rather than the surface states and Tg. During the preparation of the nano-B suspension, the nanoparticles should be 31 ACS Paragon Plus Environment


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formed from DMSO droplets in which both GLB and HPMC were dissolved. Therefore, more HPMC could be distributed not only on the surface but also throughout the nanoparticle.

Figure 9. Powder X-ray diffraction (PXRD) patterns and TEM images of (a, c) nano-A and (b, d) nano-B. The storage period is shown on the top left of each PXRD pattern. The TEM images were obtained after 4 weeks' storage at 25 ºC and 60% RH.


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Table 3. Total surface energies (γs) and their dispersive (γsD) and specific components (γsP) as calculated from IGC measurements. γs [mJ/m2] γsD [mJ/m2] γsP[mJ/m2] Amorphous GLB

70.9

29.5

41.4

HPMC

420.7

26.6

394.1

Nano-A

208.8

34.0

174.7

Nano-B

210.2

34.4

175.8

Proposed mechanism of formation of amorphous GLB nanoparticles On the basis of each analysis, the formation mechanism of nano-A and nano-B by the antisolvent method are discussed (Figure 10). In the nano-A system, the diffusion of DMSO into water was faster than that of drug molecules into the droplets. The precipitation of GLB occurred rapidly at the interface between the DMSO droplets and water. Subsequently, the aqueous solution infiltrated the particle simultaneously with the diffusion of DMSO, resulting in the formation of hollow nanoparticles (indicated by Figures 3-5).51,52 At the interface where the particles were formed, HPMC in the aqueous solution was incorporated into the GLB nanoparticles (indicated by Figure 7 and Table 1). Consequently, HPMC was localized near the particle surface (indicated by Figure 8 and Tables 2 and 3). In the nano-B system, the diffusion of DMSO into water was delayed because of the



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presence of HPMC in DMSO, and the drug molecules were able to diffuse into the droplets, accompanying the diffusion of DMSO into water. As a result, the particles became smaller and non-spherical because of partial precipitation (indicated by Figures 3-5).53,54 In addition, more HPMC was distributed inside the nanoparticles as well as on the surface in nano-B compared to nano-A, because both GLB and HPMC were dissolved in DMSO (indicated by Figures 7 and 8 and Tables 1–3). In recent years, many studies have reported the observation of liquid–liquid or glass– liquid phase separation when the drug concentration exceeds the amorphous solubility limit in an aqueous solution.55–57 The nano-A and nano-B particles were also obtained by phase separation, considering the much higher concentration of GLB (1 mg/mL) used in this study relative to the solubility of crystalline GLB (around several micrograms per milliliter).58 The nano-A and nano-B particles dispersed in the aqueous solution could be either in a supercooled liquid state above the Tg or in a glassy state below Tg depending on the temperature. The Tg of amorphous GLB is relatively high (72.8 °C, shown in Table 2) compared to those of other pharmaceutical compounds with low molecular weights.59 The Tg confinement effect would reduce the Tg of nano-A and nano-B in the aqueous solution because water, which has a low viscosity, is reported to enhance glass dynamics in a similar 34 ACS Paragon Plus Environment

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manner to air.60 However, the HPMC both at the surface and inner core of the nanoparticles should suppress the decline in Tg in the aqueous solution as well as in the solid state.43 In addition, water containment should be considered when discussing the Tg of a material dispersed in water. The Tg can be reduced by the presence of a few percent of water, because the Tg of water is quite low, at −138 °C.61 It has been reported that the Tg of pure amorphous telaprevir prepared by melt quenching at 103 °C decreases by 51 °C compared to that of ultracentrifuged precipitates of 300-nm amorphous telaprevir particles at 52 °C56 in a hydroxypropyl methylcellulose acetate succinate solution. This reduction in Tg is explained by the presence of a few percent of water. Thus, the Tg of nano-A and nano-B in an aqueous solution could be further decreased compared to those of nano-A and nano-B in the solid state because of the presence of water. Nevertheless, from the discussion of the Tg of amorphous GLB, nano-A, and nano-B in solids, we cannot determine whether nano-A and nano-B dispersed in an aqueous solution at a particular temperature are in a supercooled liquid or glassy state. Alternatively, we suggest that the characteristic hollow (nano-A) or non-spherical (nano-B) shape could be a rough but simple criterion to judge whether the amorphous nanoparticles dispersed in the aqueous solution are in a supercooled liquid or glassy state. 35 ACS Paragon Plus Environment


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Fluidic supercooled liquids form spherical droplets to reduce their surface energy, in the same way as emulsion droplets do. In reality, the shapes of amorphous nanoparticles of drugs with relatively low Tg are spherical, as revealed in cryo-TEM images.62 Meanwhile, such a morphological conversion into a spherical particle is kinetically suppressed in rigid glasses and the characteristic particle shapes are retained. The characteristic shapes of both nano-A and nano-B were maintained after storage at 4 °C for 24 h (Figure 2). The storage temperature of 4 °C is below the Tg of both nano-A and nano-B, so these particles in a rigid glass should retain their characteristic structures for a long time. On the other hand, we cannot determine if the nano-A and nano-B particles at the preparation temperature of 20– 25 °C were in a glassy state rather than a supercooled liquid state, because the viscosity of supercooled liquid states around Tg might be sufficient to form the characteristic shape of the nanoparticles. Furthermore, we must pay attention to the differences between the surface and inner core of the nanoparticles, where the molecular states and amounts of GLB and HPMC are quite different. That is, the coexistence of supercooled liquid and glassy states within a nanoparticle is possible around Tg.43 We are now investigating the effects of changing the preparation and storage temperature to discuss the relationship between the


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molecular state of the drug and polymer (supercooled liquid or glassy state) and the nanoparticle morphology.

Figure 10. Schematic representation of the precipitation process and cross-sectional structure of particles of (a) nano-A and (b) nano-B.

Conclusions Nanoparticles of amorphous GLB were successfully prepared by the antisolvent method using HPMC as a stabilizer. The morphology and stability of nano-A and nano-B were



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varied by using different methods of HPMC addition. We suggest that the diffusion rate of the solvent determines the morphology and stability of the amorphous nanoparticles prepared by the antisolvent method. The amorphous GLB in nano-B was highly stable; hence, nano-B could be applicable to various formulations. On the other hand, nano-A has a unique hollow structure, although the stability of amorphous GLB was lower than that in nano-B. In this study, the structures of amorphous GLB nanoparticles were evaluated at the molecular level by various analytical methods. Subsequently, the formation and stabilization mechanisms of amorphous nanoparticles by the antisolvent method were clarified. It is suggested that amorphous nanoparticle morphology is an indication of the molecular state (supercooled liquid or glass) of the amorphous nanoparticles in an aqueous solution, although further investigations in this regard are necessary. The findings of this study are expected to help the development of drug amorphous nanoparticle formulations, which is still investigated by a trial-and-error approach.

AUTHOR INFORMATION

Corresponding Author 38 ACS Paragon Plus Environment

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*E-mail: [email protected]

Author Contributions † H. Yonashiro and K. Higashi contributed equally as the first authors.

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENTS

We thank Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan) for gifting HPMC. This research was partly supported by a grant from the Takeda Science Foundation and a JSPS KAKENHI Grant-in-Aid for Scientific Research (C) 15K07885.

SUPPORTING INFORMATION

Zeta potentials of nano-A and nano-B suspensions (Table S1); Specific surface areas of amorphous GLB, nano-A, and nano-B (Table S2); A SEM image of amorphous GLB (Figure



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S1); Particle size distribution patterns of nano-A and nano-B as determined from FE-TEM images (Figure S2); Full solid-state 13C CP/MAS NMR spectra of crystalline GLB, HPMC, amorphous GLB, nano-A, and nano-B (Figure S3); Full solution-state 1H NMR spectra of GLB, HPMC, nano-A, and nano-B dissolved in DMSO (Figure S4). This material is available free of charge on the internet at http://pubs.acs.org/.


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Graphical abstract


Morphological and physicochemical evaluation of two distinct

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