Development and Evaluation of Docetaxel-Phospholipid Complex Loaded Self-Microemulsifying Drug Delivery System: Optimization and In Vitro/Ex Vivo Studies

Docetaxel (DTX) has clinical efficacy in the treatment of breast cancer, but it is difficult to develop a product for oral administration, due to low solubility and permeability. This study focused on preparing a self-microemulsifying drug delivery system (SME) loaded with DTX-phospholipid complex (DTX@PLC), to improve the dissolution and gastrointestinal (GI) permeability of DTX. A dual technique combining the phospholipid complexation and SME formulation described as improving upon the disadvantages of DTX has been proposed. We hypothesized that the complexation of DTX with phospholipids can improve the lipophilicity of DTX, thereby increasing the affinity of the drug to the cell lipid membrane, and simultaneously improving permeability through the GI barrier. Meanwhile, DTX@PLC-loaded SME (DTX@PLC-SME) increases the dissolution and surface area of DTX by forming a microemulsion in the intestinal fluid, providing sufficient opportunity for the drug to contact the GI membrane. First, we prepared DTX@PLC-SME by combining dual technologies, which are advantages for oral absorption. Next, we optimized DTX@PLC-SME with nanosized droplets (117.1 nm), low precipitation (8.9%), and high solubility (33.0 mg/g), which formed a homogeneous microemulsion in the aqueous phase. Dissolution and cellular uptake studies demonstrated that DTX@PLC-SME showed 5.6-fold higher dissolution and 2.3-fold higher DTX uptake in Caco-2 cells than raw material. In addition, an ex vivo gut sac study confirmed that DTX@PLC-SME improved GI permeability of DTX by 2.6-fold compared to raw material. These results suggested that DTX@PLC-SME can significantly overcome the disadvantages of anticancer agents, such as low solubility and permeability.


Screening of Mass Ratio for DTX@PLC
The ratio of DTX and phospholipid was screened to establish the optimal ratio of DTX@PLC [3]. Briefly, DTX@PLCs with different ratios (DTX:phospholipid = 1:1-5 mass ratio) were prepared with the described method. n-hexane containing 3 v/v% ethanol was added into the excess amount of DTX@PLC, and the mixture was rotated to extract DTX from the complex at room temperature for 24 h. After that, the mixture was centrifuged at 15,000 g for 5 min, and the supernatant was filtered using a 0.45 µ m polyvinylidene fluoride (PVDF) membrane filter (Millipore; Bengaluru, Karnataka, India). The filtrate was collected and diluted with 50 v/v% methanol. The dissolved amount of DTX was analyzed using HPLC. Solubility enhancing capacity per unit quantity (SEC) was calculated with the following equation [4]: where SOLDTX@PLC and SolDTX represent the solubility of DTX@PLC and DTX material, respectively, Massphospholipid represents the amount (mg) of added phospholipid.

Fourier Transform Infrared Spectroscopy (FT-IR)
In order to clarify the chemical interaction between DTX and phospholipid, Fourier transform infrared spectroscopy (FT-IR) spectra of DTX (raw material), phospholipid, physical mixture, and DTX@PLC were investigated with Thermo Scientific Nicolet 380 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The spectrum was collected in the wavelength range of 4000-500 cm −1 . Figure S1 shows that the solubility of DTX increased to 1.4 mg/mL as the mass ratio of phospholipid increased. As the phospholipid formed a complex with DTX, the solubility of DTX was improved. On the other hand, SEC showed the highest value at a mass ratio of 1:3, which means that the efficiency of DTX@PLC was the best at the mass ratio. Therefore, a mass ratio of 1:3 was chosen for further studies. Figure. S2A showed the X-ray diffraction (XRD) patterns of DTX (raw material), phospholipid, physical mixture, and DTX@PLC. The diffraction pattern of raw material showed sharp crystalline peaks at 8.8°, 11.1°, 14.0°, and 17.7°. In the case of phospholipid, the crystalline peaks at 5.8° and 7.6°. Most of the peaks were also detected in physical mixture. However, peaks indicating crystallinity were not detected in DTX@PLC, suggesting that the crystal form of DTX and phospholipid was transformed into an amorphous/solubilized form through complexation.

Physicochemical Characterizations of DTX@PLC
The chemical interaction between DTX and phospholipid was investigated using FT-IR. The FT-IR spectra of DTX (raw material), phospholipid, physical mixture, and DTX@PLC were depicted in Figure. S2B. DTX exhibited characteristic peaks at 1710 and 707 cm -1 . Phospholipid showed specific peaks at 2923, 2854, 1734, and 1062 cm -1 . In DTX@PLC, overlapped peaks of DTX and phospholipid were observed without spectral shift, indicating no interaction between DTX and phospholipid. The overall spectrum of DTX@PLC was similar to that of physical mixture. These results indicated that the functional group of DTX was invariant by complexation, suggesting that DTX could be complexed with phospholipids while maintaining the original characteristics of DTX.  Note: df (Degree of freedom), a large F-value implies a large impact on the modeling profile; a p-value of less than 0.05 means that it affects modeling profile. Note: df (Degree of freedom), a large F-value implies a large impact on the modeling profile; a p-value of less than 0.05 means that it affects modeling profile. Note: df (Degree of freedom), a large F-value implies a large impact on the modeling profile; a p-value of less than 0.05 means that it affects modeling profile. Note: df (Degree of freedom), a large F-value implies a large impact on the modeling profile; a p-value of less than 0.05 means that it affects modeling profile.

Results
To investigate the mechanism of drug release, various dissolution models (zero-order, firstorder, Higuchi, Hixson-Crowell, and Korsmeyer-Peppas models) were fitted to the release profiles of the formulations. Table S6 provides the R 2 values for all dissolution conditions tested, and the drug release mechanism was classified according to this value. As shown in the R 2 values, Korsmeyer-Peppas model fits well with the dissolution profiles of DTX@PLC, DTX-SME, and DTX@PLC-SME. In the case of DTX, most of the media, except for distilled water, followed Higuchi model. Table S7. IC50 values of docetaxel (DTX), docetaxel-phospholipid complex (DTX@PLC), docetaxelloaded self-microemulsifying drug delivery system (DTX-SME), and docetaxel-phospholipid complex loaded self-microemulsifying drug delivery system (DTX@PLC-SME), determined by MTT assay. Values are represented as mean ± SD (n = 6).

Long-term stability
The stability of DTX@PLC-SME was investigated for 4 weeks. DTX@PLC-SME was stored in glass vials capped at 25 °C for 4 weeks. The samples (100 mg) were added to 10 mL of distilled water, and then dispersed to form a homogeneous emulsion. At predetermined time-points, physical properties (particle size, precipitation, and drug content) were evaluated.

Freeze-thaw cycle stability
To test the freeze-thaw cycle stability, three freeze-thaw cycles between -20 and 25 °C were performed on DTX@PLC-SME, stored at each temperature for at least 24 h. The samples (100 mg) were added to 10 mL of distilled water and then dispersed to form a homogeneous emulsion. At predetermined time-points, physical properties (particle size, precipitation, and drug content) were evaluated.

Heating-cooling cycle stability
The cycle of storing for more than 24 h at 4 to 45 °C was studied three times. The samples (100 mg) were added to 10 mL of distilled water and then dispersed to form a homogeneous emulsion. At predetermined time-points, physical properties (particle size, precipitation, and drug content) were evaluated.