Characterization and Stability of Tanshinone IIA Solid Dispersions with Hydroxyapatite

Solid dispersions of tanshinone IIA (TanIIA) using hydroxyapatite (HAp) as the dispersing carrier (TanIIA-HAp SDs) were prepared by the solvent evaporation method. The formed solid dispersions were characterized by scanning electron microscopy (SEM), differential scanning calorimetry analysis (DSC), X-ray powder diffraction (XRPD) and Fourier transforms infrared (FTIR) spectroscopy. The in vitro dissolution rate and the stability of TanIIA-HAp SDs were also evaluated. DSC and XRPD showed that TanIIA was changed from a crystalline to an amorphous form. FTIR suggested the presence of interactions between TanIIA and HAp in solid dispersions. The result of an in vitro dissolution study showed that the dissolution rate of TanIIA-HAp SDs was nearly 7.11-folds faster than free TanIIA. Data from stability studies for over one year of TanIIA-HAp SDs performed under room temperature revealed no significant differences in drug content and dissolution behavior. All these results indicated that HAp may be a promising carrier for improving the oral absorption of TanIIA.

Solid dispersion is one of the most successful techniques, which could enhance the dissolution rate of poorly aqueous soluble drugs by particle size reduction, amorphous fraction and wettability improvement [13][14][15]. The high energy state of the amorphous form confers higher solubility and improves bioavailability through increasing dissolution. Thus, the application of amorphous phases has been the subject of very intensive investigations in the pharmaceutical field [16][17][18]. However, the thermodynamic instability of this amorphous state is generally associated with the high energy state and may lead to unacceptable physical changes, such as recrystallization during storage [19,20]. Although factors governing physical stability remain controversial and complicated, it was well known that specific interactions between drug and carrier may retard this conversion to the crystalline form [21][22][23]. As reported by Wu et al. [24] and Al-Obaidi et al. [25], the hydrogen bonding between drug and carrier is responsible for drug dispersion and depresses the crystallization of drug in solid dispersions.
Hydroxyapatite (HAp) is a natural element of human hard tissues (70% of bone is made up of this organic mineral) and has been commonly studied in bone tissue engineering, owing to its good biocompatibility [26][27][28][29]. HAp is known for its bioactive (i.e., ability of forming a direct chemical bond with surrounding tissues), osteoconductive, non-toxic, non-inflammatory and non-immunogenic properties [30,31]. Furthermore HAp has been applied widely in various biomedical applications, owning to its unique functional properties of high surface-area-to-volume ratio and porosity. Its ultrafine structure, which is similar to biological apatite's, had a great impact on cell-biomaterial interaction [32][33][34]. However, HAp has not yet been reported as a carrier of solid dispersions. Therefore, TanIIA-HAp SDs were prepared by the solvent evaporation method; and they were studied by scanning electron microscopy (SEM), differential scanning calorimetry analysis (DSC), X-ray powder diffraction (XRPD) and Fourier transforms infrared (FTIR) spectroscopy, along with the in vitro dissolution rate and stability. Through this research, we expect to pave the preliminary way towards the feasibility of HAp as a carrier of solid dispersions.

Materials
TanIIA with 98% purity was purchased from the Nanjing ZeLang Medical Technology Co., Ltd. TanIIA standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HAp was supplied by Shanghai Jiang Lai Bio-Technology Co., Ltd. All reagents were of analytical grade, except methanol, which was of chromatographic grade.

Preparation of Solid Dispersions and Physical Mixtures
Solid dispersions of TanIIA with HAp in various weight ratios were prepared using the solvent evaporation method. In brief, TanIIA was dissolved in ethanol and HAp was suspended into the above solution (the weight ratio of TanIIA and HAp was 1:3, 1:5, 1:7 and 1:9). The formed suspension was evaporated under reduced pressure in a rotavapor (Buchi, Switzerland) at 45 °C. The obtained solid dispersions were further dried in a vacuum chamber (Heraeus, Germany) at room temperature for 12 h to remove the remaining ethanol. Subsequently, samples were stored in a desiccator until further analysis. Physical mixtures were prepared by mixing TanIIA with HAp, then grinding thoroughly with a mortar and pestle until homogeneous mixtures were obtained.

HPLC Analysis of TanIIA
The concentration of TanIIA in the dissolution medium was determined by using a high performance liquid chromatography (HPLC) system (Shimadzu Scientific Instrument, MD, USA), consisting of a UV detector (SPD-10A), a pump (LC-10AD) and an automatic injector (SIL-10A). The mobile phase of methanol and water (85:15, v:v) was used at a flow rate of 1.0 mL min −1 . The samples were analyzed at 270 nm and the 30 °C temperature of a Diamonsil TM RP-C18 column (250 mm × 4.6 mm, 5 μm).

In vitro Dissolution Studies
The dissolution studies were performed using the paddle method, according to the 34th edition of US Pharmacopoeia. A ZRS-8G dissolution tester (Tianjin, China) was used with 900 mL dissolution media (distilled water contained 0.5% sodium dodecyl sulfate) volume at 37 ± 1 °C and a stirring rate of 50 rpm. The samples equivalent to 5 mg TanIIA were sealed in hard gelatin capsules with a manual capsule filling machine (CapsulCN-50, Zhejiang, China) and put into the dissolution cup. At predetermined time intervals of 15, 30, 60, 90, 120 and 180 min, 5 mL of dissolution medium were withdrawn and replaced with the same medium volume. The withdrawn samples were filtrated (0.45 μm), then spectrophotometrically assayed at 270 nm. Experiments were performed in triplicate.

Differential Scanning Calorimetry (DSC)
The DSC profiles of TanIIA, HAp, physical mixtures and solid dispersions were obtained by using differential scanning calorimeter (204A/G Phoenix ® instrument, Netzsch, Germany) at a heating rate of 10 °C/min from 25 to 500 °C in a nitrogen atmosphere.

Scanning Electron Microscopy (SEM)
SEM samples were mounted on aluminum stubs and coated with a thin gold-palladium layer using an auto-fine coater unit (Jeol, JFC, Tokyo, Japan). The surface topography was analyzed with a Jeol scanning electron microscope (JSM-6360A, Tokya, Japan) operated at an acceleration voltage of 30 kV.

X-ray Powder Diffraction (XRPD)
XRPD was performed at room temperature with a X-ray diffractometer (X-pro Pan analytical, Phillips, Mumbai, India). The data were collected through primary monochromated radiation (Cu Ká1, ë =1.5406 Å), over a 2θ range of 0°-70° with a step size of 0.04 and a dwell time of 10 s per step. About 200 mg of each sample powder were side-loaded in a sample holder to minimize possible preferential orientation.

Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopic analysis was carried out using a Nicolet Nextus 470 FTIR spectrometer (Thermo Electron Corporation, USA). The infrared spectra of the samples were recorded in the solid state using the KBr disc method over a wave number range of 4000-400 cm −1 . Individual HAp, TanIIA and physical mixtures were run as controls.

Stability Test
The prepared solid dispersions were stored for 12 months at 25 ± 2 °C and 60% ± 5% relative humidity in an artificial climate box. The extent of dissolution was analyzed at predetermined time intervals of 0, 3, 6, 9, 12 months.

In vitro Dissolution Study
The dissolution rate of TanIIA from different samples is provided in Figure 1. TanIIA crystals exhibited a low dissolution rate, with a 13.3% release in 60 min and only reaching 27.3% in 180 min, while the dissolution rate of TanIIA from the physical mixture had no significant difference compared with the free TanIIA. Furthermore, as we expected, an apparent trend was observed between the HAp ratios and the dissolution rate of TanIIA. The 1:9 SDs released 94.6% of drug in 60min, whereas 1:3, 1:5 and 1:7 SDs exhibited a release of 40.9%, 66.3% and 92.3%, respectively. Moreover, 1:7 and 1:9 SDs showed similar release curves and higher dissolution rates than the 1:3 and 1:5 SDs. These indicated that 7-9 folds of HAp carrier was enough for TanIIA SDs. In view of the need of long-term stability for SDs, 1:9 ratio SDs were chosen for further study.
There were several factors explaining the improved drug dissolution from solid dispersions, such as the enhancement in wettability and dispersibility of TanIIA, as well as the decrease in particle size. The presence of amorphous TanIIA might be a significant factor, which was confirmed by the results obtained from SEM, DSC and XRPD. HAp is a good adsorbent for a wide range of ions, small molecules and macromolecules, owing to its unique functional properties of high surface-area-to-volume ratio [35,36]. Additionally, adsorption onto insoluble, porous, high surface-area carriers is a well-known technique to enhance drug dissolution and has already been described for silica-based excipients in the early 1970s [37].

X-ray P
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Stability
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