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IJP 13990 1–7 International Journal of Pharmaceutics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Encapsulation of poorly water-soluble drugs into organic nanotubes for improving drug dissolution Kunikazu Moribe a, *, Takashi Makishima a , Kenjirou Higashi a , Nan Liu a , Waree Limwikrant b , Wuxiao Ding c , Mitsutoshi Masuda c, Toshimi Shimizu c, Keiji Yamamoto a a
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri Ayudhya Road, Ratchatewi, Bangkok 10400, Thailand Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 5, Higashi 1-1-1, Tsukuba, Ibaraki 3058565, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2013 Received in revised form 14 March 2014 Accepted 3 April 2014 Available online xxx
Hydrocortisone (HC), a poorly water-soluble drug, was encapsulated within organic nanotubes (ONTs), which were formed via the self-assembly of N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl]dodecanyl}glycylglycylglycine acid. The stability of the ONTs was evaluated in ten organic solvents, of differing polarities, by field emission transmission electron microscopy. The ONTs maintained their stable tubular structure in the highly polar solvents, such as ethanol and acetone. Furthermore, solution-state 1H-NMR spectroscopy confirmed that they were practically insoluble in acetone at 25 C (0.015 mg/mL). HCloaded ONTs were prepared by solvent evaporation using acetone. A sample with a 3/7 weight ratio of HC/ ONT was analyzed by powder X-ray diffraction, which confirmed the presence of a halo pattern and the absence of any crystalline HC peak. HC peak broadening, observed by solid-state 13C-NMR measurements of the evaporated sample, indicated the presence of HC crystals. These results indicated that HC was successfully encapsulated in ONT as an amorphous state. Improvements of the HC dissolution rate were clearly observed in aqueous media at both pH 1.2 and 6.8, probably due to HC amorphization in the ONTs. Phenytoin, another poorly water-soluble drug, also showed significant dissolution improvement upon ONT encapsulation. Therefore, ONTs can serve as an alternative pharmaceutical excipient to enhance the bioavailability of poorly water-soluble drugs. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Organic nanotube Drug encapsulation Amorphous Dissolution rate enhancement Hydrocortisone Phenytoin
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1. Introduction
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Various pharmaceutical formulations incorporate excipients such as cyclodextrin (Brewster and Loftsson, 2007; Carrier et al., 2007), surfactant (Pongpeerapat et al., 2006; Wanawongthai et al., 2009), and water-soluble polymers (Kojima et al., 2012) to improve the solubility of poorly water-soluble drugs (Yamamoto et al., 2011). Mesoporous materials have also been employed for the preparation of solid dispersions, in which drugs can be distributed into host materials. Zeolite (Braschi et al., 2010), silica xerogel (Braschi et al., 2010), calcium carbonate (Wang et al., 2006), and folded mesoporous porous silica (Nishiwaki et al., 2009) are also known as host materials for small-molecule incorporation owing to their mesoporous structure. Drug
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* Corresponding author at: Tel.: +81 43 226 2866; fax: +81 43 226 2867. E-mail address: [email protected] (N. Liu).
incorporation into the pores of the host is typically achieved by evaporation, supercritical fluid, or sealed-heating methods. Drug amorphization by incorporation into mesoporous materials resulted in enhanced dissolution characteristics and improved pharmacokinetics (Wang et al., 2009). However, mesoporous materials, such as those including metallic nuclei like Si and Al, cannot be readily used as pharmaceutical excipients owing to their unfavorable toxicological properties (Di Pasqua et al., 2008). Organic nanotubes (ONTs) are hollow cylindrical nanomaterials composed of monomeric units containing both hydrophilic and hydrophobic functionalities. The unique structures of these amphiphilic molecules drive their self-assembly in aqueous media (Masuda and Shimizu, 2004; Shimizu et al., 2005). Processes for the large-scale synthesis of amphiphiles from natural materials, such as fatty acids and glucoses, and an efficient method to induce their self-assembly into ONTs were recently disclosed (Asakawa et al., 2008). The resulting ONTs showed no toxicity in acute oral toxicity tests using rat models. Furthermore, biodegradation tests
http://dx.doi.org/10.1016/j.ijpharm.2014.04.005 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
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using environmental microorganisms revealed negligible influence of ONTs on the environment. As expected, these desirable properties have garnered considerable interest into the design and development of novel ONTs as next-generation biomaterials in various fields, especially in pharmaceutical and medical sciences. ONTs can encapsulate larger guest molecules than conventional organic host materials such as cyclodextrins can, owing to their much larger pore size (ranging from several nanometers to 100 nm). Encapsulation is typically achieved by the simple incubation of the host and guest materials in aqueous media. Indeed, the encapsulation of proteins (Kameta et al., 2008) and DNA (Ding et al., 2011; Meilander et al., 2003) has been achieved via this method. The process is driven by the combination of capillary action and electrostatic attraction. Ding et al. and Wakasugi et al. recently reported the loading of doxorubicin, an amphipathic anticancer drug, into ONTs (Ding et al., 2012a; Wakasugi et al., 2011). In this study, pH changes were utilized to affect the release of doxorubicin from the ONTs. The frequency of reports describing the loading of water-soluble drugs into ONTs is also increasing. Although the application of ONTs to encase amphipathic and water-soluble substrates is known, an efficient and reliable method for the loading of poorly water-soluble drugs into ONTs has not yet been reported. Drug encapsulation in inorganic porous materials using organic solvents has been rather successful (Nishiwaki et al., 2009); however, it is not universally applicable to ONTs because their structure is not stable in certain organic solvents. Herein, we report the application of self-assembled ONTs derived from the amphiphilic N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl]dodecanyl}-glycylglycylglycine acid (Fig. 1a) for the encapsulation of the hydrophobic drugs, hydrocortisone (HC) and phenytoin (PHE). This amphiphile possesses terminal glucose- and triglycine-headgroups to facilitate self-assembly. The resulting ONTs consist of a single monolayer lined with polyglycine-II-type hydrogen-bond networks among the triglycine moieties (Kameta et al., 2007). Therefore, the ONTs have orthogonal inner and outer surfaces adorned with polar carboxylic acid group and neutral glycosyl moieties, respectively. These ONTs demonstrate favorable
Fig. 1. Molecular structure of (a) N-{12-[(2-a,b-D-glucopyranosyl)carbamoyl] dodecanyl}-glycylglycylglycine acid, (b) hydrocortisone (HC), and (c) phenytoin (PHE).
dispersion properties in water owing to the presence of a cylindrically arranged monolayer membrane structure with the hydrophilic hydroxyl groups facing the solvent. Various solvents were examined in order to identify solvents in which the tubular structure of the ONTs is maintained. This study identified acetone as the optimal solvent for the encapsulation of poorly watersoluble drugs via solvent evaporation. The encapsulation of HC was confirmed by powder X-ray diffraction (PXRD) and solid-state 13CNMR spectroscopy. Finally, the dissolution behavior of HC from the ONTs in an aqueous medium, was evaluated.
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2. Materials and methods
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2.1. Materials
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The amphiphilic molecule, (N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl]dodecanyl}-glycylglycylglycine acid), containing glucose and triglycine groups at both ends, was synthesized as reported by Ding et al. (Ding et al., 2012b). Reagent grade HC and PHE were purchased from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan) and Nacalai Tesque, Inc. (Tokyo, Japan), respectively. The chemical structures of both HC and PHE are shown in Fig. 1.
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2.2. ONT assembly
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The amphiphile was first pulverized by mortar and pestle. The resulting material was dispersed in distilled water at a concentration of 1.0 mg/mL via sonication. The resulting suspension was then refluxed at 100 C for 10 min. At this point, the amphiphile was completely dissolved and the solution was homogenous. Next, the ONTs were precipitated by slowly cooling the solution to room
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Fig. 2. Organic nanotubes (ONTs). (a) A schematic illustration of the molecular packing of ONTs resulting from the self-assembly of the amphiphile in water. TEM images of ONT (b) negatively stained with phosphotungstate and (c) treated with acetone.
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Size dimension (nm)
Outer diameter
Inner diameter
Wall thicknessa
14.4 1.7
7.3 0.9
3.4 0.7
a
Thickness was evaluated by measuring the length of the ONT wall. The subtraction value of inner diameter from outer diameter is not always consistent with twice the value of the wall thickness.
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temperature. The mixture was frozen and water was removed by lyophilization to afford the solid ONTs (Freeze dryer FD-1000, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). 2.3. Preparation of physical mixture (PM) and evaporated sample (EVP)
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Drugs (HC or PHE) were mixed with the freshly prepared ONTs at different weight ratios of drug/ONT (3/7 and 5/5) in a glass vial for 1 min to obtain a PM. The ONTs were dispersed into an acetone solution where the drug was completely dissolved with the 3/7 and 5/5 weight ratios of drug/ONT. The obtained suspension was sonicated for 3 min and evaporated at 30 C to dryness. The resultant powder was further dried at 60 C for 24 h to obtain EVP.
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2.4. Powder X-ray diffraction experiments
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PXRD patterns were obtained using a MiniFlex II (Rigaku, Tokyo, Japan) instrument. The X-ray generator was operated at 30 kV and 15 mA, using CuKa radiation. The scans were performed between 2 and 35 with a scanning rate of 4 (2u)/min at room temperature.
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2.5. Field emission transmission electron microscopy (FE-TEM) experiments
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The ONT dispersions in aqueous solutions or organic solvents were dropped onto a grid supported by either butyral film (STEM Cu160, Oken Shoji Co., Ltd., Tokyo, Japan) or Excel Support Film1 (Cu 200, Nisshin EM Co., Ltd., Tokyo, Japan), respectively. The ONT was permitted to adsorb onto the grids for 5 min before the excess solution was removed by blotting with a filter paper. Negative staining was performed for 1 min using a 2% w/v phosphotungstate solution (pH 7.0). Finally, the grid was dried in a desiccator at room temperature for 24 h prior to FE-TEM analysis at 120 kV using JEOLJEM2100F (JEOL Ltd., Tokyo, Japan).
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2.6. Solution-state 1H-NMR measurements
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All 1H-NMR spectra were acquired using an ECA-500 NMR spectrometer (JEOL RESONANCE Co., Ltd., Tokyo, Japan) with a
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magnetic field of 11.7 T. 1H-NMR samples were prepared by dispersing the ONTs into dimethylsulfoxide-d6 and acetone-d6 at a concentration of 1.0 mg/mL via sonication. 1H-NMR spectra were recorded at 25 C using a relaxation delay of 60 s and 32 accumulations. Chemical shifts were referenced to the internal standard signal of 0.05% tetramethylsilane (TMS) at 0 ppm. The amphiphilic molecule peak was integrated against that of TMS in order to determine the degree of ONT solubility in acetone.
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2.7. Solid-state NMR measurements
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All solid-state 13C-NMR spectra were obtained using an ECA600 NMR spectrometer (JEOL RESONANCE Co., Ltd., Tokyo, Japan) with a magnetic field of 14.1 T. Powdered samples (ca. 150 mg) were placed in a 4 mm zirconium rotor. The 13C spectra were acquired using cross-polarization (CP) together with magic angle spinning (MAS) at 15 kHz and high-power 1H decoupling at an inlet air temperature of 25 C. For each spectrum, the total number of accumulations (1000–10,000) was dependent upon the required signal-to-noise ratio. Pertinent acquisition parameters included relaxation delays of 4–7 s, a CP contact time of 5 ms, and a 90 1Hpulse of 2.7 s. A total of 2048 data points were collected per spectrum in each experiment and zero-filled to 8192 points. All spectra were externally referenced to TMS by setting the methine peak of hexamethylbenzene to 17.3 ppm.
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2.8. Dissolution tests
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Dissolution tests were carried out using a dissolution test apparatus NTR-VS6P (Toyama Sangyo Co., Ltd., Osaka, Japan) in accordance with the Japanese Pharmacopeia (JP) XVI paddle method. The dissolution profiles of the powdered samples were obtained in 500 mL of JP 1st fluid (pH 1.2) and 2nd fluid (pH 6.8). The paddle rotational speed was set at 50 and 100 rpm for HC and PHE system, respectively. A constant temperature bath was maintained at 37.0 0.5 C. The dissolution experiment was initiated by placing the sample in the dissolution vessel. Sample aliquots of 5 mL were withdrawn at specific intervals (3, 5, 10, 20, 30, 60, and 120 min) with dissolution medium replacement. The aliquots were filtered through a 0.20 mm cellulose nitrate membrane prior to analysis with HPLC. The HPLC conditions were as follows: Capcell Pak C18 SG-120 column (Shiseido Co., Ltd., Tokyo, Japan), a flow rate of 1.0 mL/min, and a constant column temperature of 40 C. Absorbance was recorded at 245 nm for HC and 258 nm for PHE using a UV–vis detector. The mobile phase was composed of water:acetonitrile:tetrahydrofuran = 60:30:10 (v/v/v) for HC and methanol:0.01 M phosphate buffer (pH 3.0) = 55:45 (v/ v) for PHE.
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Table 2 Appearance of ONTs dispersed in various solvents and their morphology observed by TEM. Solvent
Dielectric constant
Appearance of ONT dispersed in solvent
ONT tube structure observed by TEM
n-Hexane Cyclohexane Diethylether Chloroform Dichloromethane 2-Propanol Acetone Ethanol Methanol N,N-dimethylformamide Acetonitrile Dimethylsulfoxide Water
1.88 2.02 4.33 4.81 8.93 18.3 20.7 25.8 32.7 36.7 37.5 46.7 81.1
aggregation aggregation aggregation aggregation aggregation suspension suspension suspension suspension clear solution suspension clear solution suspension
x x x x x – –
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Table 3 Peak integral value and concentration of TMS and ONT in acetone at 25 C (n = 3, mean SD). Sample (chemical shift) TMS (0.00 ppm) ONT (1.28 ppm) a
Peak integral value 1.000 0.009
Concentration (mg/mL) a
0.324 0.015 0.002
Initial concentration.
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3. Results and discussion
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3.1. Formation of ONTs from amphiphile
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FE-TEM observation clearly confirmed that the asymmetric bolaamphiphile, N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl] dodecanyl}-glycylglycylglycine acid, self-assembled into ONTs in water (Fig. 2b). Table 1 shows the average inner and outer diameters, and the membrane thickness of the ONTs, which were determined using 250 randomly-selected pieces of ONTs on the FE-TEM micrographs. The small size distribution, with respect to ONT length, revealed that the monolayer membranes were uniformly formed.
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3.2. Structural stability of ONT in various organic solvents
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The structural stability of ONTs suspended in organic solvents was evaluated in order to select a suitable solvent for drug encapsulation by evaporation. ONTs were reconstituted into various solvents at a concentration of 1.0 mg/mL prior to sonication. The resultant ONT-dispersed solutions are shown in Fig. S1. Clear solutions were obtained when the ONTs were dispersed into the highly polar solvents, N,N-dimethylformamide and dimethylsulfoxide, indicating that the ONTs readily dissolved in both solvents. In contrast, ONT agglomeration was observed in nhexane, cyclohexane, diethylether, chloroform, and dichloromethane. However, ONTs were homogeneously dispersed when they were formed in 2-propanol, acetone, ethanol, methanol, acetonitrile, and water. The ONT structures, dispersed into organic solvents, were thoroughly evaluated by FE-TEM (Fig. 2c and
Fig. S2). These data indicate that the tubular structures are maintained in 2-propanol, acetone (Fig. 2c), ethanol, methanol, acetonitrile, and water. The dielectric constant is considered as an index of a solvent’s polarity because it is proportional to the dipole moment of an organic solvent. Table 2 summarizes the appearance, morphology, and dielectric constant of solutions containing ONTs for each solvent. The results of this study clearly indicate a correlation between the polarity of the organic solvent and ONT stability, except N,N-dimethylformamide and dimethylsulfoxide which can dissolve ONTs. Taken together with the FE-TEM data, it can be said that ONTs can be homogeneously dispersed in highly polar solvents, such as 2-propanol, acetone, ethanol, methanol, and acetonitrile because they can maintain their structures in these solvents. Among the high polarity solvents which retained the tubular structure, acetone has the lowest boiling point. As such, it was chosen as the solvent for drug encapsulation by evaporation. With the optimal solvent identified, 1H-NMR measurements were performed to evaluate ONT solubility in acetone. Fig. S3 shows the 1H-NMR spectra of ONT dispersed in acetone-d6 and dimethylsulfoxide-d6. While the 1H-NMR peaks of the ONTs were very small in acetone, they were all clearly observed in dimethylsulfoxide. The integral value of the ONT oligomethylene group (1.28 ppm) as compared to that of methyl group in the internal standard, TMS (0.00 ppm), was determined. This value was utilized to calculate the concentration of the dissolved ONTs in acetone. As shown in Table 3, the ONT solubility in acetone was determined to be 0.015 0.002 mg/mL, while that in dimethylsulfoxide was 1.0 mg/mL. It was confirmed that ONTs can be homogeneously dispersed into highly polar solvents without dissolution and without perturbing their tubular structure. Agglomeration behavior of ONT in relatively lower dielectric solvents can be explained as follows. The ONT that is covered by hydrophilic functional groups, can be easily dispersed into high polar solvents. Hydration of the hydrophilic functional groups, hydrophilic interaction between them, and hydrophobic interaction between acyl chains should be driving forces for selfassembling of ONT dispersed in aqueous media. On the other hand, the hydrogen-bond networks can be broken by changing dielectric condition in lower dielectric solvents. In this case, the amphiphile may be aggregated by transforming from ONT.
Fig. 3. Powder X-ray diffraction (PXRD) patterns of HC/ONT and PHE/ONT systems: (a) HC, (b) ONT, (c) HC/ONT 3/7 PM, (d) HC/ONT 3/7 EVP, (e) HC/ONT 5/5 EVP, (f) PHE, (g) ONT, (h) PHE/ONT 3/7 PM, (i) PHE/ONT 3/7 EVP, and (j) PHE/ONT 5/5 EVP. (&) HC, (*) PHE. The ratios indicate weight ratios.
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Fig. 4. 13C-CP/MAS NMR spectra of (a) HC, (b) ONT, and (c) HC/ONT 3/7 EVP. Asterisk (*) shows the spinning side bands. The numbers in the spectrum correspond to the carbons in the HC structure (Fig. 1b).
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3.3. HC encapsulation into ONTs The encapsulation of HC into ONTs was achieved by evaporation method using acetone. The crystalline state of the samples was evaluated by PXRD (Fig. 3). While diffraction peaks at 2u = 14.4 and 17.4 (Fig. 3a) are characteristic of crystalline HC, the ONTs produced a broad diffraction peak at around 2u = 21 (Fig. 3b). In HC/ONT PM, at a weight ratio of 3/7, diffraction peaks of HC crystals were observed (Fig. 3c). In contrast, those peaks disappeared and a halo pattern was observed in the EVP (Fig. 3d). This indicates that HC transforms from crystalline to an amorphous state in the presence of ONT that are prepared via solvent evaporation. For HC/ ONT 5/5 EVP, diffraction peaks of HC crystals were still observed (Fig.3e). From this data one can conclude that the maximum amount of HC that is loaded into ONTs is approximately 30%. It is expected that encapsulation efficiency of a drug changes depending on structures of the amphiphile and the self-assembled ONT. Inner diameter of ONT also affects the encapsulation efficiency. Drug-encapsulated amount depends on specific surface area of
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ONT pore and the volume. According to our knowledge using mesoporous silica with different pore diameters, the maximum encapsulation efficiency should exist in each drug/mesoporous silica system and is usually about 30–40% depending on the combination of the materials. In this study, encapsulation efficiency was about 30% which appeared to be close to the maximum encapsulation efficiency. Next, the molecular state of HC in EVP was investigated by solid-state 13C-NMR(Fig. 4, Table S1). HC showed characteristic peaks at 200–220 ppm and 120–180 ppm, which are derived from its carbonyls and carbon–carbon double bond, respectively (Yang et al., 2008). In the spectrum of ONT, the peaks derived from the amide and carboxylic acid carbonyls were observed between 170– 180 ppm. The NMR peak width is a reflection of molecular mobility, thus the broadening of 13C-NMR peaks is indicative of a suppression or restriction of mobility. In the EVP spectrum, the carbon–carbon double bond peak of HC (C5) was difficult to evaluate because of the overlap with the carbonyl peaks. Peak broadenings of HC, e.g., C3, C4, and C20 were also observed as compared with those of crystalline HC. This result indicates that HC changed its molecular state from crystalline to amorphous during the evaporation process. Molecular structure and the twodimensional molecular length of HC (14.3 Å 16.1 Å) based on van der Waals radius of carbon atom are shown in Fig. 1b. The inner diameter of the ONTs (7.3 nm, Table 1) was found to be approximately five times that of the length of HC. SliwinskaBartkowiak et al. reported the effect of pore diameter of mesoporous silica on guest drug amorphization (SliwinskaBartkowiak et al., 2001). In this report, the authors mentioned that amorphization of drugs in the pores occurred when pore diameters of host were 15 times larger than the size of the guest. This supported our findings that HC in EVP was encapsulated in the amorphous state in the ONTs. The TEM images of the ONTs, which were produced by sonication in acetone, indicated a degree of secondary structure i.e., they were gathered in bundles and stacked on top of each other. Kameta et al. reported that ONTs possess not only cylindrical inner spaces, but also interstitial spaces formed among the ONT cylinders (Kameta et al., 2011). They also mentioned that materials could be captured into both the inner and outer spaces of the ONTs. Furthermore, it has also been reported that ONTs can encapsulate macromolecules such as proteins within both their hollow cylindrical interior as well as on their external three-dimensional network (Kameta et al., 2009). As such, it was estimated that HC could be encapsulated within both the hollow nanospace and the interstitial exterior space of ONTs without crystallization.
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3.4. Dissolution behavior of poorly water-soluble drugs encapsulated within ONTs
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Fig. 5a and b show the dissolution profiles of HC encapsulated within ONTs under sink condition at pH 6.8 and pH 1.2, respectively. The solubility of HC at pH 6.8 and pH 1.2 remains relatively constant (0.32 and 0.33 mg/mL respectively) because it does not contain readily ionizable groups. At pH 6.8, the percentage of drug dissolved from unprocessed HC was approximately 20% at 10 min. Unprocessed HC exhibited the lowest dissolution profile. In HC/ONT PM, the percentage of dissolved HC increased to ca. 40%. This is likely due to the improved wettability of HC crystals in the presence of ONTs, which have a good dispersibility in aqueous media. On the other hand, HC in HC/ONT EVP rapidly dissolved to approximately 90% in 10 min. Thus, dissolution of HC was significantly improved by the encapsulation into ONT. Next, the dissolution profile of HC was investigated at a lower pH (pH 1.2). As expected, the dissolution behavior of HC at pH 1.2 was quite similar to that at pH 6.8. Percentages of dissolved
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Fig. 5. Dissolution profiles of HC/ONT and PHE/ONT systems using a dissolution test apparatus according to the Japanese Pharmacopeia (JP) XVI paddle method at 37 C. (n = 3, mean SD) (a) HC/ONT system in JP 2nd fluid (pH 6.8); (&) HC, (4) HC/ONT 3/7 PM, and () HC/ONT 3/7 EVP, (b) HC/ONT system in JP 1st fluid (pH 1.2); (&) HC, (~) HC/ONT 3/7 PM, and (*) HC/ONT 3/7 EVP, and (c) PHE/ONT system in JP 2nd fluid (pH 6.8); ($) PHE, (5) PHE/ONT 3/7 PM, and (^) PHE/ONT 3/7 EVP. Samples of 8.0 mg (26.7 mg of EVP) and 8.3 mg (27.7 mg of EVP) of HC were added to 500 mL of JP 2nd and 1st fluids, respectively. 5 mg (16.7 mg of EVP) of PHE was added to JP 2nd fluid.
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HC from unprocessed HC, HC/ONT PM, and EVP at 10 min were approximately 20, 40, and 90%, respectively. The pH-independent dissolution of HC should be due to the non-ionizable property of HC under the conditions evaluated. The dissolution of drug/ONT EVP was further evaluated using another poorly water-soluble drug, PHE. The physicochemical properties of PHE/ONT EVP were evaluated by PXRD (Figs. 3f–j). Diffraction peaks of PHE crystals were observed in PHE/ONT 3/7 PM, whereas in the case of 3/7 EVP those peaks were replaced with those corresponding to a halo pattern. As with HC, PHE transformed from crystalline to amorphous state upon evaporation with ONT. For PHE/ONT 5/5 EVP, diffraction peaks of PHE crystals were observed. Thus, the maximum amount of encapsulated PHE within ONT was approximately 30%. The dissolution profile of PHE from ONT under sink condition at pH 6.8 is shown in Fig. 5c. As expected, the solubility of PHE at pH 6.8 was 0.14 mg/mL, which was almost identical to that at pH 1.2 (0.13 mg/mL). At pH 6.8, unprocessed PHE apparently showed the lowest percentage of dissolution, as the dissolved PHE was less than 5% after 10 min. In PHE/ONT PM, the degree of PHE dissolution increased to ca. 20%. On the other hand, approximately 70% of the PHE in PHE/ONT EVP dissolved after 10 min. When compared with the unprocessed PHE and PHE/ONT PM, a significant enhancement in the dissolution of PHE was
achieved in PHE/ONT EVP. These results indicate that the observed dissolution improvements using ONT may be applicable for other poorly water-soluble drugs. As shown in Figs. 3 and 4, amorphization of the drug in the presence of ONT could be a key to the improved drug dissolution. The amorphous form possesses higher energy than the crystalline form, which is a driving force for the enhancement of drug dissolution. A similar dissolution improvement by amorphization was reported using drug/mesoporous silica system (Tozuka et al., 2003).
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4. Conclusions
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In this study, the encapsulation of poorly water-soluble drugs, HC and PHE, into ONTs was achieved by evaporation method using acetone. The encapsulated drug was found to exist in an amorphous state in the hollow nanospace and the interstitial space of the ONT. The dissolution rates of the drugs from ONTs were clearly improved as compared to that of the unprocessed drug and PM at pH 6.8. A pH-independent dissolution was observed using non-ionizable substrates. The results strongly suggest that ONTs can be used as a useful host material for the pharmaceutical formulation of poorly water-soluble drugs.
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Acknowledgements
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This study was partly supported by Grants-in-Aid for Research on Development of New Drugs from the Japan Health Sciences Foundation and, fundings for Scientific Research (C) (JSPS, 24590045, 25460032) and for Young Scientist (B) (JSPS, 24790041) from the Japan Society for the Promotion of Sciences.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.04.005.
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Asakawa, M., Aoyagi, M., Kameta, N., Kogiso, M., Masuda, M., Minamikawa, H., Shimizu, T., 2008. Development of massive synthesis method of organic nanotube toward practical use. Synthesiology 1, 183–189 translated. Braschi, I., Gatti, G., Paul, G., Gessa, C.E., Cossi, M., Marchese, L., 2010. Sulfonamide antibiotics embedded in high silica zeolite Y: a combined experimental and rheoretical study of host–guest and guest–guest interactions. Langmuir 26, 9524–9532. Brewster, M.E., Loftsson, T., 2007. Cyclodextrins as pharmaceutical solubilizers. Advanced Drug Delivery Reviews 59, 645–666. Carrier, R.L., Miller, L.A., Ahmed, I., 2007. The utility of cyclodextrins for enhancing oral bioavailability. Journal of Controlled Release 123, 78–99. Di Pasqua, A.J., Sharma, K.K., Shi, Y.L., Toms, B.B., Ouellette, W., Dabrowiak, J.C., Asefa, T., 2008. Cytotoxicity of mesoporous silica nanomaterials. Journal of Inorganic Biochemistry 102, 1416–1423. Ding, W., Wada, M., Kameta, N., Minamikawa, H., Shimizu, T., Masuda, M., 2011. Functionalized organic nanotubes as tubular nonviral gene transfer vector. Journal of Controlled Release 156, 70–75. Kameta, N., Minamikawa, H., Masuda, M., 2011. Supramolecular organic nanotubes: how to utilize the inner nanospace and the outer space. Soft Matter 7, 4539–4561. Ding, W., Kameta, N., Minamikawa, H., Wada, M., Shimizu, T., Masuda, M., 2012a. Hybrid organic nanotubes with dual functionalities localized on cylindrical nanochannels control the release of doxorubicin. Advanced Healthcare Materials 1, 699–706. Ding, W., Wada, M., Minamikawa, H., Kameta, N., Masuda, M., Shimizu, T., 2012b. Cisplatin-encapsulated organic nanotubes by endo-complexation in the hollow cylinder. Chemical Communications 48, 8625–8627. Kameta, N., Mizuno, G., Masuda, M., Minamikawa, H., Kogiso, M., Shimizu, T., 2007. Molecular monolayer nanotubes having 7–9 nm inner diameters covered with different inner and outer surfaces. Chemistry Letters 36, 896–897.
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Kameta, N., Minamikawa, H., Mizuno, G., Masuda, M., Shimizu, T., 2008. Controllable biomolecule release from self-assembled organic nanotubes with asymmetric surfaces: pH and temperature dependence. Soft Matter 4, 1681–1687. Kameta, N., Yoshida, K., Masuda, M., Shimizu, T., 2009. Supramolecular nanotube hydrogels: remarkable resistance effect of confined proteins to denaturants. Chemistry of Materials 21, 5892–5898. Kojima, T., Higashi, K., Suzuki, T., Tomono, K., Moribe, K., Yamamoto, K., 2012. Stabilization of a supersaturated solution of mefenamic acid from a solid dispersion with EUDRAGIT1 EPO. Pharmaceutical Research 1–15. Masuda, M., Shimizu, T., 2004. Lipid nanotubes and microtubes: experimental evidence for unsymmetrical monolayer membrane formation from unsymmetrical bolaamphiphiles. Langmuir 20, 5969–5977. Meilander, N.J., Pasumarthy, M.K., Kowalczyk, T.H., Cooper, M.J., Bellamkonda, R.V., 2003. Sustained release of plasmid DNA using lipid microtubules and agarose hydrogel. Journal of Controlled Release 88, 321–331. Nishiwaki, A., Watanabe, A., Higashi, K., Tozuka, Y., Moribe, K., Yamamoto, K., 2009. Molecular states of prednisolone dispersed in folded sheet mesoporous silica (FSM-16). International Journal of Pharmaceutics 378, 17–22. Pongpeerapat, A., Higashi, K., Tozuka, Y., Moribe, K., Yamamoto, K., 2006. Molecular interaction among probucol/PVP/SDS multicomponent system investigated by solid-state NMR. Pharmaceutical Research 23, 2566–2574. Shimizu, T., Masuda, M., Minamikawa, H., 2005. Supramolecular nanotube architectures based on amphiphilic molecules. Chemical Reviews 105, 1401–1444. Sliwinska-Bartkowiak, M., Dudziak, G., Gras, R., Sikorski, R., Radhakrishnan, R., Gubbins, K.E., 2001. Freezing behavior in porous glasses and MCM-41. Colloids and Surfaces A: Physiological and Engineering Aspects 187, 523–529. Tozuka, Y., Oguchi, T., Yamamoto, K., 2003. Adsorption and entrapment of salicylamide molecules into the mesoporous structure of folded sheets mesoporous material (FSM-16). Pharmaceutical Research 20, 926–930. Wanawongthai, C., Pongpeerapat, A., Higashi, K., Tozuka, Y., Moribe, K., Yamamoto, K., 2009. Nanoparticle formation from probucol/PVP/sodium alkyl sulfate coground mixture. International Journal of Pharmaceutics 376, 169–175. Wakasugi, A., Asakawa, M., Kogiso, M., Shimizu, T., Sato, M., Maitani, Y., 2011. Organic nanotubes for drug loading and cellular delivery. International Journal of Pharmaceutics 413, 271–278. Wang, C., He, C., Tong, Z., Liu, X., Ren, B., Zeng, F., 2006. Combination of adsorption by porous CaCO3 microparticles and encapsulation by polyelectrolyte multilayer films for sustained drug delivery. International Journal of Pharmaceutics 308, 160–167. Wang, F., Hui, H., Barnes, T.J., Barnett, C., Prestidge, C.A., 2009. Oxidized mesoporous silicon microparticles for improved oral delivery of poorly soluble drugs. Molecular Pharmaceutics 7, 227–236. Yamamoto, K., Limwikrant, W., Moribe, K., 2011. Analysis of molecular interactions in solid dosage forms; challenge to molecular pharmaceutics. Chemical and Pharmaceutical Bulletin 59, 147–154. Yang, J.H., Ho, Y., Tzou, D.L.M., 2008. A 13C solid state NMR analysis of steroid compounds. Magnetic Resonance in Chemistry 46, 718–725.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Encapsulation of poorly water-soluble drugs into organic nanotubes for improving drug dissolution Kunikazu Moribe a, *, Takashi Makishima a , Kenjirou Higashi a , Nan Liu a , Waree Limwikrant b , Wuxiao Ding c , Mitsutoshi Masuda c, Toshimi Shimizu c, Keiji Yamamoto a a
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri Ayudhya Road, Ratchatewi, Bangkok 10400, Thailand Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 5, Higashi 1-1-1, Tsukuba, Ibaraki 3058565, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2013 Received in revised form 14 March 2014 Accepted 3 April 2014 Available online xxx
Hydrocortisone (HC), a poorly water-soluble drug, was encapsulated within organic nanotubes (ONTs), which were formed via the self-assembly of N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl]dodecanyl}glycylglycylglycine acid. The stability of the ONTs was evaluated in ten organic solvents, of differing polarities, by field emission transmission electron microscopy. The ONTs maintained their stable tubular structure in the highly polar solvents, such as ethanol and acetone. Furthermore, solution-state 1H-NMR spectroscopy confirmed that they were practically insoluble in acetone at 25 C (0.015 mg/mL). HCloaded ONTs were prepared by solvent evaporation using acetone. A sample with a 3/7 weight ratio of HC/ ONT was analyzed by powder X-ray diffraction, which confirmed the presence of a halo pattern and the absence of any crystalline HC peak. HC peak broadening, observed by solid-state 13C-NMR measurements of the evaporated sample, indicated the presence of HC crystals. These results indicated that HC was successfully encapsulated in ONT as an amorphous state. Improvements of the HC dissolution rate were clearly observed in aqueous media at both pH 1.2 and 6.8, probably due to HC amorphization in the ONTs. Phenytoin, another poorly water-soluble drug, also showed significant dissolution improvement upon ONT encapsulation. Therefore, ONTs can serve as an alternative pharmaceutical excipient to enhance the bioavailability of poorly water-soluble drugs. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Organic nanotube Drug encapsulation Amorphous Dissolution rate enhancement Hydrocortisone Phenytoin
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1. Introduction
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Various pharmaceutical formulations incorporate excipients such as cyclodextrin (Brewster and Loftsson, 2007; Carrier et al., 2007), surfactant (Pongpeerapat et al., 2006; Wanawongthai et al., 2009), and water-soluble polymers (Kojima et al., 2012) to improve the solubility of poorly water-soluble drugs (Yamamoto et al., 2011). Mesoporous materials have also been employed for the preparation of solid dispersions, in which drugs can be distributed into host materials. Zeolite (Braschi et al., 2010), silica xerogel (Braschi et al., 2010), calcium carbonate (Wang et al., 2006), and folded mesoporous porous silica (Nishiwaki et al., 2009) are also known as host materials for small-molecule incorporation owing to their mesoporous structure. Drug
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* Corresponding author at: Tel.: +81 43 226 2866; fax: +81 43 226 2867. E-mail address: [email protected] (N. Liu).
incorporation into the pores of the host is typically achieved by evaporation, supercritical fluid, or sealed-heating methods. Drug amorphization by incorporation into mesoporous materials resulted in enhanced dissolution characteristics and improved pharmacokinetics (Wang et al., 2009). However, mesoporous materials, such as those including metallic nuclei like Si and Al, cannot be readily used as pharmaceutical excipients owing to their unfavorable toxicological properties (Di Pasqua et al., 2008). Organic nanotubes (ONTs) are hollow cylindrical nanomaterials composed of monomeric units containing both hydrophilic and hydrophobic functionalities. The unique structures of these amphiphilic molecules drive their self-assembly in aqueous media (Masuda and Shimizu, 2004; Shimizu et al., 2005). Processes for the large-scale synthesis of amphiphiles from natural materials, such as fatty acids and glucoses, and an efficient method to induce their self-assembly into ONTs were recently disclosed (Asakawa et al., 2008). The resulting ONTs showed no toxicity in acute oral toxicity tests using rat models. Furthermore, biodegradation tests
http://dx.doi.org/10.1016/j.ijpharm.2014.04.005 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
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using environmental microorganisms revealed negligible influence of ONTs on the environment. As expected, these desirable properties have garnered considerable interest into the design and development of novel ONTs as next-generation biomaterials in various fields, especially in pharmaceutical and medical sciences. ONTs can encapsulate larger guest molecules than conventional organic host materials such as cyclodextrins can, owing to their much larger pore size (ranging from several nanometers to 100 nm). Encapsulation is typically achieved by the simple incubation of the host and guest materials in aqueous media. Indeed, the encapsulation of proteins (Kameta et al., 2008) and DNA (Ding et al., 2011; Meilander et al., 2003) has been achieved via this method. The process is driven by the combination of capillary action and electrostatic attraction. Ding et al. and Wakasugi et al. recently reported the loading of doxorubicin, an amphipathic anticancer drug, into ONTs (Ding et al., 2012a; Wakasugi et al., 2011). In this study, pH changes were utilized to affect the release of doxorubicin from the ONTs. The frequency of reports describing the loading of water-soluble drugs into ONTs is also increasing. Although the application of ONTs to encase amphipathic and water-soluble substrates is known, an efficient and reliable method for the loading of poorly water-soluble drugs into ONTs has not yet been reported. Drug encapsulation in inorganic porous materials using organic solvents has been rather successful (Nishiwaki et al., 2009); however, it is not universally applicable to ONTs because their structure is not stable in certain organic solvents. Herein, we report the application of self-assembled ONTs derived from the amphiphilic N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl]dodecanyl}-glycylglycylglycine acid (Fig. 1a) for the encapsulation of the hydrophobic drugs, hydrocortisone (HC) and phenytoin (PHE). This amphiphile possesses terminal glucose- and triglycine-headgroups to facilitate self-assembly. The resulting ONTs consist of a single monolayer lined with polyglycine-II-type hydrogen-bond networks among the triglycine moieties (Kameta et al., 2007). Therefore, the ONTs have orthogonal inner and outer surfaces adorned with polar carboxylic acid group and neutral glycosyl moieties, respectively. These ONTs demonstrate favorable
Fig. 1. Molecular structure of (a) N-{12-[(2-a,b-D-glucopyranosyl)carbamoyl] dodecanyl}-glycylglycylglycine acid, (b) hydrocortisone (HC), and (c) phenytoin (PHE).
dispersion properties in water owing to the presence of a cylindrically arranged monolayer membrane structure with the hydrophilic hydroxyl groups facing the solvent. Various solvents were examined in order to identify solvents in which the tubular structure of the ONTs is maintained. This study identified acetone as the optimal solvent for the encapsulation of poorly watersoluble drugs via solvent evaporation. The encapsulation of HC was confirmed by powder X-ray diffraction (PXRD) and solid-state 13CNMR spectroscopy. Finally, the dissolution behavior of HC from the ONTs in an aqueous medium, was evaluated.
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2. Materials and methods
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2.1. Materials
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The amphiphilic molecule, (N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl]dodecanyl}-glycylglycylglycine acid), containing glucose and triglycine groups at both ends, was synthesized as reported by Ding et al. (Ding et al., 2012b). Reagent grade HC and PHE were purchased from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan) and Nacalai Tesque, Inc. (Tokyo, Japan), respectively. The chemical structures of both HC and PHE are shown in Fig. 1.
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2.2. ONT assembly
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The amphiphile was first pulverized by mortar and pestle. The resulting material was dispersed in distilled water at a concentration of 1.0 mg/mL via sonication. The resulting suspension was then refluxed at 100 C for 10 min. At this point, the amphiphile was completely dissolved and the solution was homogenous. Next, the ONTs were precipitated by slowly cooling the solution to room
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Fig. 2. Organic nanotubes (ONTs). (a) A schematic illustration of the molecular packing of ONTs resulting from the self-assembly of the amphiphile in water. TEM images of ONT (b) negatively stained with phosphotungstate and (c) treated with acetone.
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Size dimension (nm)
Outer diameter
Inner diameter
Wall thicknessa
14.4 1.7
7.3 0.9
3.4 0.7
a
Thickness was evaluated by measuring the length of the ONT wall. The subtraction value of inner diameter from outer diameter is not always consistent with twice the value of the wall thickness.
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temperature. The mixture was frozen and water was removed by lyophilization to afford the solid ONTs (Freeze dryer FD-1000, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). 2.3. Preparation of physical mixture (PM) and evaporated sample (EVP)
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Drugs (HC or PHE) were mixed with the freshly prepared ONTs at different weight ratios of drug/ONT (3/7 and 5/5) in a glass vial for 1 min to obtain a PM. The ONTs were dispersed into an acetone solution where the drug was completely dissolved with the 3/7 and 5/5 weight ratios of drug/ONT. The obtained suspension was sonicated for 3 min and evaporated at 30 C to dryness. The resultant powder was further dried at 60 C for 24 h to obtain EVP.
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2.4. Powder X-ray diffraction experiments
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PXRD patterns were obtained using a MiniFlex II (Rigaku, Tokyo, Japan) instrument. The X-ray generator was operated at 30 kV and 15 mA, using CuKa radiation. The scans were performed between 2 and 35 with a scanning rate of 4 (2u)/min at room temperature.
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2.5. Field emission transmission electron microscopy (FE-TEM) experiments
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The ONT dispersions in aqueous solutions or organic solvents were dropped onto a grid supported by either butyral film (STEM Cu160, Oken Shoji Co., Ltd., Tokyo, Japan) or Excel Support Film1 (Cu 200, Nisshin EM Co., Ltd., Tokyo, Japan), respectively. The ONT was permitted to adsorb onto the grids for 5 min before the excess solution was removed by blotting with a filter paper. Negative staining was performed for 1 min using a 2% w/v phosphotungstate solution (pH 7.0). Finally, the grid was dried in a desiccator at room temperature for 24 h prior to FE-TEM analysis at 120 kV using JEOLJEM2100F (JEOL Ltd., Tokyo, Japan).
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2.6. Solution-state 1H-NMR measurements
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All 1H-NMR spectra were acquired using an ECA-500 NMR spectrometer (JEOL RESONANCE Co., Ltd., Tokyo, Japan) with a
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magnetic field of 11.7 T. 1H-NMR samples were prepared by dispersing the ONTs into dimethylsulfoxide-d6 and acetone-d6 at a concentration of 1.0 mg/mL via sonication. 1H-NMR spectra were recorded at 25 C using a relaxation delay of 60 s and 32 accumulations. Chemical shifts were referenced to the internal standard signal of 0.05% tetramethylsilane (TMS) at 0 ppm. The amphiphilic molecule peak was integrated against that of TMS in order to determine the degree of ONT solubility in acetone.
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2.7. Solid-state NMR measurements
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All solid-state 13C-NMR spectra were obtained using an ECA600 NMR spectrometer (JEOL RESONANCE Co., Ltd., Tokyo, Japan) with a magnetic field of 14.1 T. Powdered samples (ca. 150 mg) were placed in a 4 mm zirconium rotor. The 13C spectra were acquired using cross-polarization (CP) together with magic angle spinning (MAS) at 15 kHz and high-power 1H decoupling at an inlet air temperature of 25 C. For each spectrum, the total number of accumulations (1000–10,000) was dependent upon the required signal-to-noise ratio. Pertinent acquisition parameters included relaxation delays of 4–7 s, a CP contact time of 5 ms, and a 90 1Hpulse of 2.7 s. A total of 2048 data points were collected per spectrum in each experiment and zero-filled to 8192 points. All spectra were externally referenced to TMS by setting the methine peak of hexamethylbenzene to 17.3 ppm.
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2.8. Dissolution tests
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Dissolution tests were carried out using a dissolution test apparatus NTR-VS6P (Toyama Sangyo Co., Ltd., Osaka, Japan) in accordance with the Japanese Pharmacopeia (JP) XVI paddle method. The dissolution profiles of the powdered samples were obtained in 500 mL of JP 1st fluid (pH 1.2) and 2nd fluid (pH 6.8). The paddle rotational speed was set at 50 and 100 rpm for HC and PHE system, respectively. A constant temperature bath was maintained at 37.0 0.5 C. The dissolution experiment was initiated by placing the sample in the dissolution vessel. Sample aliquots of 5 mL were withdrawn at specific intervals (3, 5, 10, 20, 30, 60, and 120 min) with dissolution medium replacement. The aliquots were filtered through a 0.20 mm cellulose nitrate membrane prior to analysis with HPLC. The HPLC conditions were as follows: Capcell Pak C18 SG-120 column (Shiseido Co., Ltd., Tokyo, Japan), a flow rate of 1.0 mL/min, and a constant column temperature of 40 C. Absorbance was recorded at 245 nm for HC and 258 nm for PHE using a UV–vis detector. The mobile phase was composed of water:acetonitrile:tetrahydrofuran = 60:30:10 (v/v/v) for HC and methanol:0.01 M phosphate buffer (pH 3.0) = 55:45 (v/ v) for PHE.
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Table 2 Appearance of ONTs dispersed in various solvents and their morphology observed by TEM. Solvent
Dielectric constant
Appearance of ONT dispersed in solvent
ONT tube structure observed by TEM
n-Hexane Cyclohexane Diethylether Chloroform Dichloromethane 2-Propanol Acetone Ethanol Methanol N,N-dimethylformamide Acetonitrile Dimethylsulfoxide Water
1.88 2.02 4.33 4.81 8.93 18.3 20.7 25.8 32.7 36.7 37.5 46.7 81.1
aggregation aggregation aggregation aggregation aggregation suspension suspension suspension suspension clear solution suspension clear solution suspension
x x x x x – –
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Table 3 Peak integral value and concentration of TMS and ONT in acetone at 25 C (n = 3, mean SD). Sample (chemical shift) TMS (0.00 ppm) ONT (1.28 ppm) a
Peak integral value 1.000 0.009
Concentration (mg/mL) a
0.324 0.015 0.002
Initial concentration.
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3. Results and discussion
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3.1. Formation of ONTs from amphiphile
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FE-TEM observation clearly confirmed that the asymmetric bolaamphiphile, N-{12-[(2-a,b-D-glucopyranosyl) carbamoyl] dodecanyl}-glycylglycylglycine acid, self-assembled into ONTs in water (Fig. 2b). Table 1 shows the average inner and outer diameters, and the membrane thickness of the ONTs, which were determined using 250 randomly-selected pieces of ONTs on the FE-TEM micrographs. The small size distribution, with respect to ONT length, revealed that the monolayer membranes were uniformly formed.
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3.2. Structural stability of ONT in various organic solvents
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The structural stability of ONTs suspended in organic solvents was evaluated in order to select a suitable solvent for drug encapsulation by evaporation. ONTs were reconstituted into various solvents at a concentration of 1.0 mg/mL prior to sonication. The resultant ONT-dispersed solutions are shown in Fig. S1. Clear solutions were obtained when the ONTs were dispersed into the highly polar solvents, N,N-dimethylformamide and dimethylsulfoxide, indicating that the ONTs readily dissolved in both solvents. In contrast, ONT agglomeration was observed in nhexane, cyclohexane, diethylether, chloroform, and dichloromethane. However, ONTs were homogeneously dispersed when they were formed in 2-propanol, acetone, ethanol, methanol, acetonitrile, and water. The ONT structures, dispersed into organic solvents, were thoroughly evaluated by FE-TEM (Fig. 2c and
Fig. S2). These data indicate that the tubular structures are maintained in 2-propanol, acetone (Fig. 2c), ethanol, methanol, acetonitrile, and water. The dielectric constant is considered as an index of a solvent’s polarity because it is proportional to the dipole moment of an organic solvent. Table 2 summarizes the appearance, morphology, and dielectric constant of solutions containing ONTs for each solvent. The results of this study clearly indicate a correlation between the polarity of the organic solvent and ONT stability, except N,N-dimethylformamide and dimethylsulfoxide which can dissolve ONTs. Taken together with the FE-TEM data, it can be said that ONTs can be homogeneously dispersed in highly polar solvents, such as 2-propanol, acetone, ethanol, methanol, and acetonitrile because they can maintain their structures in these solvents. Among the high polarity solvents which retained the tubular structure, acetone has the lowest boiling point. As such, it was chosen as the solvent for drug encapsulation by evaporation. With the optimal solvent identified, 1H-NMR measurements were performed to evaluate ONT solubility in acetone. Fig. S3 shows the 1H-NMR spectra of ONT dispersed in acetone-d6 and dimethylsulfoxide-d6. While the 1H-NMR peaks of the ONTs were very small in acetone, they were all clearly observed in dimethylsulfoxide. The integral value of the ONT oligomethylene group (1.28 ppm) as compared to that of methyl group in the internal standard, TMS (0.00 ppm), was determined. This value was utilized to calculate the concentration of the dissolved ONTs in acetone. As shown in Table 3, the ONT solubility in acetone was determined to be 0.015 0.002 mg/mL, while that in dimethylsulfoxide was 1.0 mg/mL. It was confirmed that ONTs can be homogeneously dispersed into highly polar solvents without dissolution and without perturbing their tubular structure. Agglomeration behavior of ONT in relatively lower dielectric solvents can be explained as follows. The ONT that is covered by hydrophilic functional groups, can be easily dispersed into high polar solvents. Hydration of the hydrophilic functional groups, hydrophilic interaction between them, and hydrophobic interaction between acyl chains should be driving forces for selfassembling of ONT dispersed in aqueous media. On the other hand, the hydrogen-bond networks can be broken by changing dielectric condition in lower dielectric solvents. In this case, the amphiphile may be aggregated by transforming from ONT.
Fig. 3. Powder X-ray diffraction (PXRD) patterns of HC/ONT and PHE/ONT systems: (a) HC, (b) ONT, (c) HC/ONT 3/7 PM, (d) HC/ONT 3/7 EVP, (e) HC/ONT 5/5 EVP, (f) PHE, (g) ONT, (h) PHE/ONT 3/7 PM, (i) PHE/ONT 3/7 EVP, and (j) PHE/ONT 5/5 EVP. (&) HC, (*) PHE. The ratios indicate weight ratios.
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Fig. 4. 13C-CP/MAS NMR spectra of (a) HC, (b) ONT, and (c) HC/ONT 3/7 EVP. Asterisk (*) shows the spinning side bands. The numbers in the spectrum correspond to the carbons in the HC structure (Fig. 1b).
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3.3. HC encapsulation into ONTs The encapsulation of HC into ONTs was achieved by evaporation method using acetone. The crystalline state of the samples was evaluated by PXRD (Fig. 3). While diffraction peaks at 2u = 14.4 and 17.4 (Fig. 3a) are characteristic of crystalline HC, the ONTs produced a broad diffraction peak at around 2u = 21 (Fig. 3b). In HC/ONT PM, at a weight ratio of 3/7, diffraction peaks of HC crystals were observed (Fig. 3c). In contrast, those peaks disappeared and a halo pattern was observed in the EVP (Fig. 3d). This indicates that HC transforms from crystalline to an amorphous state in the presence of ONT that are prepared via solvent evaporation. For HC/ ONT 5/5 EVP, diffraction peaks of HC crystals were still observed (Fig.3e). From this data one can conclude that the maximum amount of HC that is loaded into ONTs is approximately 30%. It is expected that encapsulation efficiency of a drug changes depending on structures of the amphiphile and the self-assembled ONT. Inner diameter of ONT also affects the encapsulation efficiency. Drug-encapsulated amount depends on specific surface area of
5
ONT pore and the volume. According to our knowledge using mesoporous silica with different pore diameters, the maximum encapsulation efficiency should exist in each drug/mesoporous silica system and is usually about 30–40% depending on the combination of the materials. In this study, encapsulation efficiency was about 30% which appeared to be close to the maximum encapsulation efficiency. Next, the molecular state of HC in EVP was investigated by solid-state 13C-NMR(Fig. 4, Table S1). HC showed characteristic peaks at 200–220 ppm and 120–180 ppm, which are derived from its carbonyls and carbon–carbon double bond, respectively (Yang et al., 2008). In the spectrum of ONT, the peaks derived from the amide and carboxylic acid carbonyls were observed between 170– 180 ppm. The NMR peak width is a reflection of molecular mobility, thus the broadening of 13C-NMR peaks is indicative of a suppression or restriction of mobility. In the EVP spectrum, the carbon–carbon double bond peak of HC (C5) was difficult to evaluate because of the overlap with the carbonyl peaks. Peak broadenings of HC, e.g., C3, C4, and C20 were also observed as compared with those of crystalline HC. This result indicates that HC changed its molecular state from crystalline to amorphous during the evaporation process. Molecular structure and the twodimensional molecular length of HC (14.3 Å 16.1 Å) based on van der Waals radius of carbon atom are shown in Fig. 1b. The inner diameter of the ONTs (7.3 nm, Table 1) was found to be approximately five times that of the length of HC. SliwinskaBartkowiak et al. reported the effect of pore diameter of mesoporous silica on guest drug amorphization (SliwinskaBartkowiak et al., 2001). In this report, the authors mentioned that amorphization of drugs in the pores occurred when pore diameters of host were 15 times larger than the size of the guest. This supported our findings that HC in EVP was encapsulated in the amorphous state in the ONTs. The TEM images of the ONTs, which were produced by sonication in acetone, indicated a degree of secondary structure i.e., they were gathered in bundles and stacked on top of each other. Kameta et al. reported that ONTs possess not only cylindrical inner spaces, but also interstitial spaces formed among the ONT cylinders (Kameta et al., 2011). They also mentioned that materials could be captured into both the inner and outer spaces of the ONTs. Furthermore, it has also been reported that ONTs can encapsulate macromolecules such as proteins within both their hollow cylindrical interior as well as on their external three-dimensional network (Kameta et al., 2009). As such, it was estimated that HC could be encapsulated within both the hollow nanospace and the interstitial exterior space of ONTs without crystallization.
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3.4. Dissolution behavior of poorly water-soluble drugs encapsulated within ONTs
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Fig. 5a and b show the dissolution profiles of HC encapsulated within ONTs under sink condition at pH 6.8 and pH 1.2, respectively. The solubility of HC at pH 6.8 and pH 1.2 remains relatively constant (0.32 and 0.33 mg/mL respectively) because it does not contain readily ionizable groups. At pH 6.8, the percentage of drug dissolved from unprocessed HC was approximately 20% at 10 min. Unprocessed HC exhibited the lowest dissolution profile. In HC/ONT PM, the percentage of dissolved HC increased to ca. 40%. This is likely due to the improved wettability of HC crystals in the presence of ONTs, which have a good dispersibility in aqueous media. On the other hand, HC in HC/ONT EVP rapidly dissolved to approximately 90% in 10 min. Thus, dissolution of HC was significantly improved by the encapsulation into ONT. Next, the dissolution profile of HC was investigated at a lower pH (pH 1.2). As expected, the dissolution behavior of HC at pH 1.2 was quite similar to that at pH 6.8. Percentages of dissolved
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Fig. 5. Dissolution profiles of HC/ONT and PHE/ONT systems using a dissolution test apparatus according to the Japanese Pharmacopeia (JP) XVI paddle method at 37 C. (n = 3, mean SD) (a) HC/ONT system in JP 2nd fluid (pH 6.8); (&) HC, (4) HC/ONT 3/7 PM, and () HC/ONT 3/7 EVP, (b) HC/ONT system in JP 1st fluid (pH 1.2); (&) HC, (~) HC/ONT 3/7 PM, and (*) HC/ONT 3/7 EVP, and (c) PHE/ONT system in JP 2nd fluid (pH 6.8); ($) PHE, (5) PHE/ONT 3/7 PM, and (^) PHE/ONT 3/7 EVP. Samples of 8.0 mg (26.7 mg of EVP) and 8.3 mg (27.7 mg of EVP) of HC were added to 500 mL of JP 2nd and 1st fluids, respectively. 5 mg (16.7 mg of EVP) of PHE was added to JP 2nd fluid.
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HC from unprocessed HC, HC/ONT PM, and EVP at 10 min were approximately 20, 40, and 90%, respectively. The pH-independent dissolution of HC should be due to the non-ionizable property of HC under the conditions evaluated. The dissolution of drug/ONT EVP was further evaluated using another poorly water-soluble drug, PHE. The physicochemical properties of PHE/ONT EVP were evaluated by PXRD (Figs. 3f–j). Diffraction peaks of PHE crystals were observed in PHE/ONT 3/7 PM, whereas in the case of 3/7 EVP those peaks were replaced with those corresponding to a halo pattern. As with HC, PHE transformed from crystalline to amorphous state upon evaporation with ONT. For PHE/ONT 5/5 EVP, diffraction peaks of PHE crystals were observed. Thus, the maximum amount of encapsulated PHE within ONT was approximately 30%. The dissolution profile of PHE from ONT under sink condition at pH 6.8 is shown in Fig. 5c. As expected, the solubility of PHE at pH 6.8 was 0.14 mg/mL, which was almost identical to that at pH 1.2 (0.13 mg/mL). At pH 6.8, unprocessed PHE apparently showed the lowest percentage of dissolution, as the dissolved PHE was less than 5% after 10 min. In PHE/ONT PM, the degree of PHE dissolution increased to ca. 20%. On the other hand, approximately 70% of the PHE in PHE/ONT EVP dissolved after 10 min. When compared with the unprocessed PHE and PHE/ONT PM, a significant enhancement in the dissolution of PHE was
achieved in PHE/ONT EVP. These results indicate that the observed dissolution improvements using ONT may be applicable for other poorly water-soluble drugs. As shown in Figs. 3 and 4, amorphization of the drug in the presence of ONT could be a key to the improved drug dissolution. The amorphous form possesses higher energy than the crystalline form, which is a driving force for the enhancement of drug dissolution. A similar dissolution improvement by amorphization was reported using drug/mesoporous silica system (Tozuka et al., 2003).
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4. Conclusions
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In this study, the encapsulation of poorly water-soluble drugs, HC and PHE, into ONTs was achieved by evaporation method using acetone. The encapsulated drug was found to exist in an amorphous state in the hollow nanospace and the interstitial space of the ONT. The dissolution rates of the drugs from ONTs were clearly improved as compared to that of the unprocessed drug and PM at pH 6.8. A pH-independent dissolution was observed using non-ionizable substrates. The results strongly suggest that ONTs can be used as a useful host material for the pharmaceutical formulation of poorly water-soluble drugs.
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Acknowledgements
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This study was partly supported by Grants-in-Aid for Research on Development of New Drugs from the Japan Health Sciences Foundation and, fundings for Scientific Research (C) (JSPS, 24590045, 25460032) and for Young Scientist (B) (JSPS, 24790041) from the Japan Society for the Promotion of Sciences.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.04.005.
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