Development of a sorafenib-loaded solid self-nanoemulsifying drug delivery system: Formulation optimization and characterization of enhanced properties

Abstract

Sorafenib, marketed under the brand name Nexavar^®, is a multiple tyrosine kinase inhibitor drug that has been actively used in the clinical setting for the treatment of several cancers. However, the low solubility and bioavailability of sorafenib constitute a significant barrier to achieving a good therapeutic outcome. We developed a sorafenib-loaded self-nanoemulsifying drug delivery system (SNEDDS) formulation composed of capmul MCM, tween 80, and tetraglycol, and demonstrated that the SNEDDS formulation could improve drug solubility with excellent self-emulsification ability. Moreover, the sorafenib-loaded SNEDDS exhibited anticancer activity against Hep3B and KB cells, which are the most commonly used hepatocellular carcinoma and oral cancer cell lines, respectively. Subsequently, to improve the storage stability and to increase the possibility of commercialization, a solid SNEDDS for sorafenib was further developed through the spray drying method using Aerosil^® 200 and PVP K 30. X-ray diffraction and differential scanning calorimeter data showed that the crystallinity of the drug was markedly reduced, and the dissolution rate of the drug was further improved in formulation in simulated gastric and intestinal fluid conditions. In vivo study, the bioavailability of the orally administered formulation increases dramatically compared to the free drug. Our results highlight the use of the solid-SNEDDS formulation to enhance sorafenib’s bioavailability and outlines potential translational directions for oral drug development.

Graphical Abstract

1. Introduction

Primary liver cancer is a global health problem that is expected to affect more than one million people annually by 2025. The most common type is hepatocellular carcinoma (HCC), which has an increasing incidence worldwide and is mainly caused by chronic hepatitis [1, 2]. Currently, the standard treatment for HCC is sorafenib, a tyrosine kinase inhibitor (TKI). Sorafenib is a multi-target tyrosine kinase inhibitor that blocks the activity of c-Raf, b-Raf, vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR-β) [3–5]. Thus, in addition to HCC, it is also used to treat several cancers like renal cell carcinoma, and metastatic thyroid cancer [4, 6–8]. However, sorafenib is poorly soluble, metabolized rapidly in the liver, and is a known substrate for p-glycoprotein [3, 9, 10]. It, therefore, has low bioavailability when administered orally. To overcome these limitations, several types of drug delivery systems have been developed including liposomes, micelles, solid lipid nanoparticles, nanostructured lipid carriers, microemulsion, and the self-nanoemulsifying drug delivery system (SNEDDS) [11–18]. Among the drug delivery systems, there are active studies on the SNEDDS formulation for oral administration, and in particular, some studies report that the bioavailability of the drug is better when SNEDDS is used compared to solid dispersions [19, 20].

SNEDDS is a homogeneous mixture of oil, surfactant, and cosurfactant. It can form an emulsion in an aqueous phase such as in the gastrointestinal tract even with light agitation and has a particle size in the range of ≤ 200 nm when dispersed [21, 22]. Particularly, SNEDDS is known to be effective in solubilizing poorly soluble drugs; therefore, it has been applied to many drugs including sorafenib to increase their oral administration bioavailability. However, the existing liquid SNEDDS has stability concerns due to the agglomeration between particles, resulting in sedimentation or phase separation. Moreover, the limited storage conditions in soft capsules, complicated process control issues, and high manufacturing costs are additional issues to be addressed [23–26]. To overcome these limitations, SNEDDS solidification studies using methods such as spray drying, adsorptions to solid carriers, melt extrusion, melt granulation, and supercritical fluid-based methods are actively underway [23, 27–31].

Numerous techniques have been investigated for developing SNEDDS formulations that improve the bioavailability of Sorafenib [32, 33]. However, these studies have solely concentrated on liquid-SNEDDS formulations, and there is currently no research on the use of solid-SNEDDS formulations, which may enhance storage stability - an essential aspect of the productization process. In the current research, we evaluated multiple surfactants, oils, and cosolvents to determine the best formulation for Sorafenib-encapsulated SNEDDS. In addition, we used spray drying technology to create a solid SNEDDS formulation, which demonstrated better storage stability and improved bioavailability.

2. Materials and methods

2.1. Materials

Sorafenib tosylate was purchased from BIOSYNTH Carbosynth (Compton, Newbury, United Kingdom). Cremophor EL, tetraglycol, triethylamine, and polyvinylpyrrolidone (PVP K 30) (average molecular weight 40 kDa) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triacetin, isopropyl myristate (IPM), soybean oil, corn oil, tween 80, span 80, span 20, mineral oil, and glycerin were acquired from Daejung Chemicals Co. (Siheung, South Korea). Peceol, labrafil M 1994 CS, labrafil M 2125, labrasol, transcutol P, lauroglycol 90, capryol 90, and labrafac lipophile WL 1349 were obtained from Gattefossé (Saint-Priest Cedex, France). Capmul MCM and captex 355 were gifted from Abitec Corporation (Columbus, OH, USA). Propylene glycol, tween 20, olive oil, and phosphoric acid were purchased from Duksan Pure Chemical (Ansan, South Korea). Polyethylene glycol (PEG) 400 was purchased from Samchun Pure Chemical (Gyeonggi-Do, South Korea). Colloidal silica (Aerosil^® 200 Pharma) was purchased from Evonik industries AG (Essen, Germany). Ethanol (EtOH) and high-performance liquid chromatography (HPLC)-grade acetonitrile (ACN) were purchased from Honeywell Burdick & Jackson^® (Muskegon, MI, USA). All other chemicals used were of the analytical grade.

2.2. High-performance liquid chromatography (HPLC)

Quantitative determination of sorafenib concentration was performed by HPLC (Agilent 1200 series, Agilent Tech., USA) equipped with an autosampler, high-pressure gradient pump, a UV-Vis detector, and an Agilent 9 ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm, 5 mm, Agilent Tech., USA). The mobile phase was composed of DW containing 2% (w/v) triethylamine (pH 5.4 adjusted with phosphoric acid) and an acetonitrile mixture (30:70 v/v %) and was delivered isocratically. The retention time of sorafenib was 3.2 min when the flow rate was maintained at 1.0 mL/min and column oven temperature was kept at 25 °C. The injection volume was 20 μL and the flow rate was maintained at 1.0 mL/min. The column effluent was detected at 265 nm and the concentration of sorafenib was calculated based on a linear calibration curve of standard sorafenib solutions [26].

2.3. Screening solubility of sorafenib

The solubility of sorafenib in various oils, surfactants, and cosolvents was screened to select appropriate components for SNEDDS development. An excess amount of sorafenib powder (15 mg) was added to 1 mL of each excipient, followed by 30 min of vortexing and sonication. To obtain a supersaturated solution, the mixture was shaken for 24 h in a 37 °C water bath at 100 rpm, and it was frequently mixed with a shaking water bath (NEXUS technologies.co., Seoul, South Korea). After centrifuging the samples at 10,000 × g for 10 min, each of the supernatants was filtered through a 0.45 μ m polytetrafluoroethylene (PTFE) membrane injection filter (C&W Technologies, FL, USA) to remove insoluble sorafenib. The obtained solution was diluted 50-fold in the HPLC mobile phase and the concentration of sorafenib in each solution was assessed using an HPLC (Agilent 1200 series, Agilent Tech., USA) system.

2.4. Construction of the pseudo-ternary phase diagram

For the identification of the self-emulsifying region, a pseudo-ternary phase diagram was constructed by visual observation after 100-fold dilution with distilled water [34]. Based on the solubility study, capmul MCM, tween 80, and tetraglycol were selected as the oil, surfactant, and cosolvent, respectively, and were subsequently mixed with various proportions. The efficiency of nanoemulsion formation was evaluated by adding 100 μL of each mixture into 10 mL of distilled water with mild stirring at 300 rpm using a magnetic stirrer. The tendency of spontaneous emulsification was considered “good” when the droplets spread out easily in water and formed a fine emulsion without aggregation. Conversely, it was regarded as “bad” when there was poor or no emulsion formation due to the immediate coalescence of oil droplets, especially when the stirring was stopped [35]. All studies were performed in triplicate.

2.5. Preparation and characterization of SNEDDS

Preparation of drug-loaded SNEDDS

To prepare sorafenib-loaded liquid SNEDDS formulations, sorafenib powder was added to the selected nano-emulsion followed by vortexing and sonication until a clear solution was obtained. The prepared SNEDDS formulation was diluted 100-fold with distilled water and gently stirred (300 rpm). After 30 min, each formulation was filtered through a 0.45 μm PTFE membrane filter to remove unencapsulated sorafenib. The drug content was calculated using the following equation [36]:

$$\text{Drug content }\left(\%\right)=\left(\text{Weight of drug in the SNEDDS formulation}\right)/\left(\text{Weight of total ingredients in the SNEDDS formulation}\right)\times 100.$$

The concentration of sorafenib was determined by using HPLC.

Determination of droplet size and PDI

The effective hydrodynamic diameter (D_eff.) and polydispersity index (PDI) of the nano-emulsion were measured by a photon correlation spectrometer using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with the Multi-Angle Sizing Option (BI-MAS). Each sample was diluted 100-fold with distilled water before measurement. The average D_eff. and PDI were measured from three measurements performed on each sample [34].

Percentage transmittance

To evaluate the self-emulsification efficacy of selected SNEDDS formulations, the transmittance of selected nanoemulsions was examined using a UV-1200 Spectrophotometer (Labentech, Incheon, Republic of Korea). Each sample was diluted 100-fold with distilled water. The percentage transmittance as a factor determining optical clarity was measured at 650 nm using distilled water as blank [24].

In vitro characterization

Hep3B cells and KB cells were cultured in RPMI with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37 °C. For the cytotoxicity test, cells harvested from growing cell monolayers were seeded at a density of 10⁴ cells/well in 96 well plates. The next day, each sample was added to the cells flowed by 48 h incubation. Cell viability was evaluated using the CCK-8 assay. The absorbance at 450 nm of each well of the plates was recorded on a Flexstation 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA). IC 50 values were calculated using the Graphpad Prism 7.03 software. Cells were seeded at a density of 20 × 10⁴ cells per well in 24-well plates and exposed to samples for 24 h for caspase 3/7 activity measurement. The cells in plates were carefully washed with sterile phosphate-buffered saline (PBS), and Caspase-3/7 Green ReadyProbes^™ Reagent (R37111, Invitrogen) was added to each well and incubated for 30 min. The stained cells were analyzed using a Moticam Pro 205A camera coupled to a computer with the Motic Images Plus 3.0 software (Richmond, BC, Canada).

2.6. Preparation and characterization of solid SNEDDS

Preparation of solid SNEDDS

Solid SNEDDS formulations were prepared using a spray dryer (SD-1000, Eyela, Japan). Aerosil^® 200 and PVP K 30 were selected as the carriers for the solidification of liquid SNEDDS. Solid carriers were suspended in ethanol and then the sorafenib-loaded liquid SNEDDS was added to the solution under vigorous stirring conditions. The mixture was continually stirred while spray dried to maintain a stable suspension state under conditions with an inlet temperature of 100 °C, an outlet temperature of 50 °C, a drying air blower late of 0.40 m³/min, and a spray pressure of 100 kPa. The obtained solid SNEDDS was passed through a 355 μm mesh-testing sieve (Chung Gye Industrial Mfg., Co., South Korea). The morphology of the raw sorafenib powder and solid SNEDDS formulations was observed using a field emission-scanning electron microscope (FE-SEM) (Sigma, Carl Zeiss, Germany)

Solid-state characterization and thermal Analysis

X-ray diffractometer (XRD) measurements were performed using a New D8-Advance XRD instrument (Bruker-AXS, Germany) equipped with a copper anode generating a Cu K_α radiation of 1.54178 Å when struck by a 100-mA current at 40 kV. Patterns were collected using a step width of 0.02 °/s over a range from 5–50° on a 2θ scale at room temperature [37].

Differential scanning calorimeter (DSC) measurements were performed by Thermal Analysis system using a DSC Q2000 (TA Instruments, DE, USA). Each accurately weighed sample (approximately 5 mg) was placed in standard aluminum pans, and nitrogen was used as the effluent gas. All samples were scanned at a temperature ramp speed of 10 °C/min and heat flow from 20 to 260 °C [36].

Storage stability test

Sorafenib-loaded liquid or solid SNEEDS were incubated at different storage conditions (4°C, 25°C, and 37°C) for the storage stability test. At the predetermined time points, samples were collected, diluted 100-fold with distilled water, and gently stirred (300 rpm) for 30 min. Unencapsulated drugs were removed via filtration through a 0.45 μm PTFE membrane filter, and the drug contents in the solutions were analyzed using HPLC.

In vitro drug release profile

Dissolution profiles of sorafenib-loaded solid SNEDDS formulations were investigated by a USP 29 apparatus II using a dissolution tester (KDT-600, Kukje Eng., Korea). The dissolution media was kept at 37 ± 0.5 °C and the speed of the paddle was set to 100 rpm throughout the testing period. Milled powders of sorafenib and solid SNEDDS formulations containing 15 mg of sorafenib equivalent were exposed to 900mL of simulated gastric fluids (distilled water at pH 1.2) or simulated intestinal fluids (1× PBS with 2% Tween 80, pH 6.8). An aliquot (1 mL) of the sample was withdrawn at predetermined time points (5, 10, 15, 30, 45, 60, 90, and 120 min) from a dissolution medium and filtered using a 0.45 μm PTFE membrane filter. Each aliquot was analyzed by HPLC, as previously described. Measurements were done in triplicate.

In vitro transport of SNEDDS across the Caco-2 cells monolayer

Caco-2 cells were incubated in standard humidified conditions with 5% CO2 at 37 °C of RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. The transport study was conducted on the monolayer of Caco-2 cells cultured onto the polyester plates (24-well trans-well plates). Before cell seeding, polycarbonate trans-well inserts (0.3 μm pore size inserts, 0.33 cm² growth area) were coated by Geltrex. The inserts were washed with Dulbecco’s PBS twice, and then the Caco-2 cells were seeded at a density of 10 × 10⁴ cells/insert. The culture medium in the inserts was changed every 2 days for 21 days. The integrity of the cell monolayer was evaluated by measuring transepithelial electrical resistance (TEER) with the EVOM2 instrument (EVOM2^™, World Precision Instruments, USA). Monolayers with > 300 Ω· cm² TEER were used for transport analysis. Raw sorafenib or sorafenib-loaded solid SNEDDS were dispersed into the culture medium, and transport of the sorafenib in the apical to basolateral direction across the cell monolayer was performed by adding 0.5 mL (Equivalent drug concentration: 80 μg/mL) to the apical side and 1 mL full RPMI media to the basolateral side of the monolayer. We periodically withdrew 500 μL from the basolateral chambers at the predetermined time (30, 60, 120, 180, and 240 min), and replaced them with the same amount of pre-warmed full media. Three different inserts were used for each time point, and the transepithelial transport of sorafenib across cell monolayers was analyzed by HPLC.

Animal care

All animal care and experiments were performed under the protocol approved by the Institutional Animal Care and Use Committee of Chung-Ang University (Seoul, Republic of Korea) and the National Institute of Health guidelines (“Guide for the care and use of laboratory animals” and “Animal Protection Law in Republic of Korea”). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Chung-Ang University of Korea (approval number: A2022034).

In vivo pharmacokinetic study

Male Sprague Dawley rats (body weight: 250 g, Nara Biotech, Seoul, South Korea) were used in a set of in-vivo studies that were approved by the institution. A total of three rats were used in the study. The rats were orally administered with sorafenib tosylate and F22, which were dispersed in water, at a dose of 50 mg/kg using a sonde. Serial blood samples, approximately 0.5 ml each, were taken from cardiac puncture at 1, 3, 6, 9, 24, and 48 hours after administration using pre-heparinized microcentrifuge tubes. The plasma samples were then centrifuged at 13,200 rpm for 10 minutes and stored at −20°C until drug analysis. For calibration, reference sorafenib dissolved in DMSO was serially diluted and added to drug-free rat plasma to obtain final concentrations ranging from 0 to 25 μg/ml. Plasma (100 μL) and acetonitrile (400 μL) were mixed and vortexed. The mixtures were then centrifuged at 10,000 ×g for 10 min and the supernatant layers were collected. After collecting the 300 μL of supernatant, it was mixed with methanol at a 1:1 ratio and filtered through a 0.45 μm filter for further analysis. Quantitative determination of sorafenib concentration in plasma was performed by HPLC (Agilent 1200 series, Agilent Tech., USA) equipped with an autosampler, high-pressure gradient pump, a UV-Vis detector, and an Agilent 9 ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm, 5 mm, Agilent Tech., USA). Using the Phoenix WinNonlin software (Version 8.3. Princeton, NJ, USA), non-compartmental analysis was used to calculate various pharmacokinetic parameters. These included the area under the plasma concentration versus time curve from zero to 48 hours (AUC 0→48h), the elimination rate (K _el), and the half-life (t_1/2). The maximum plasma concentration (C_max) and the time required to reach C _max (T _max) were directly obtained from the plasma data.

Statistical analysis

The experimental results were expressed as means ± SD. Statistical comparison of data was performed by the unpaired two-tailed Student’s t-test (n = 3). Statistical analyses were performed using GraphPad Prism. *p < 0.05, **p < 0.01, ***p < 0.001.

3. Results

3.1. Components of SNEDDS and the pseudo-ternary phase diagram

Screening drug solubility in oils and surfactants is a prerequisite for optimizing the components of SNEDDS formulations [24]. The solubility of sorafenib in various oils, surfactants, and cosolvents is presented in Figure 1(a–c). Among the oils, sorafenib was most soluble in capmul MCM (5.16 ± 0.8 mg/mL), followed by peceol (0.9 ± 0.5 mg/mL), labrafil M 1944 CS (0.35 ± 0.05 mg/mL), lauroglycol (297.6 ± 184.4 μg/mL), carpyol 90 (278.8 ± 6.5 μg/mL), labrafil M 2125 CS (278.8 ± 44.9 μg/mL), triacetin (113.1 ± 62.5 μg/mL), soybean oil (64.0 ± 49.3 μg/mL), corn oil (53.0 ± 13.7 μ g/mL), mineral oil (47.6 ± 25.2 μ g/mL), captex 355 (42.8 ± 7.6 μ g/mL), isopropyl myristate (37.9 ± 7.2 μg/mL), and olive oil (35.0 ± 0.6 μg/mL). Since the oil component is the main reservoir of hydrophobic drugs in SNEDDS, the capmul MCM, which showed the highest drug solubilizing ability was selected as the oil phase.

Figure 1.

Solubility of sorafenib in various (a) oils, (b) 5% surfactants, and (c) cosolvents (n = 3). (d) Pseudo-ternary phase diagram of self-nanoemulsions using capmul MCM as oil, tween 80 as surfactant, and tetraglycol as cosolvent. The blue points indicate the selected nanoemulsions. (e) Composition and (f) optical images of selected nanoemulsions.

Subsequently, the solubility of sorafenib in 5 % non-ionic surfactant in water was examined. Sorafenib was most soluble in labrasol (572.7 ± 39.5 μg/mL), followed by tween 80 (530.3 ± 9.7 μg/mL), cremophor EL (510.2 ± 10.7 μg/mL), tween 20 (448.4 ± 6.4 μg/mL), glycerin (178.3 ± 66.7 μg/mL), span 80 (97.0 ± 36.9 μg/mL), and span 20 (27.4 ± 6.5 μg/mL). Labrasol and tween 80 showed similar solubilizing ability, but as tween 80 has a higher hydrophilic and lipophilic balance value than labrasol, it was selected for the surfactant component [38, 39]. When labrasol was used as a surfactant rather than tween 80, the particle size and PDI values of SNEDDS were not favorable (Figure S1). Among those examined cosolvents, sorafenib was most soluble in tetraglycol (23.3 ± 3.1 mg/mL) followed by transcutol P (19.8 ± 2.8 mg/mL), PEG 400 (18.6 ± 2.9 mg/mL), propylene glycol (14.2 ± 1.0 mg/mL), and ethanol (5.8 ± 0.8 mg/mL).

Consequently, capmul MCM, tween80, and tetraglycol were selected as the oil, surfactant, and cosolvent components respectively. Subsequently, a pseudo-ternary phase diagram was constructed to obtain an optimal component ratio (Figure 1(d)). From the pseudo-ternary phase diagram, nine different formulations were selected from the miscible self-emulsifying region (Figure 1(e)), and its optical images are shown in Figure 1(f). All formulations showed a very clear appearance without any phase separations or aggregations.

3.2. Characterization of SNEDDS formulations

The selected SNEDDS formulations were dropped into distilled water, and the droplet size, PDI, and transmittance were measured. Droplet size is an important factor in self-emulsification as it determines the rate and extent of drug release and absorption [40, 41]. As shown in Figure 2(a), the size of the nanoemulsion selected above was all about 100 nm, and the PDI also showed a value less than 0.25, indicating mono-dispersity.

Figure 2.

Characterization of blank and sorafenib-loaded SNEDDSs. (a) Particle size and PDI, and (b) transmittance of blank SNEDDS formulations. (c) Particle size and PDI, (d) drug loading content, and (e) colloidal stability of sorafenib-loaded SNEDDS formulations.

The transmittance of selected SNEDDS droplets at λ= 580 nm is shown in Figure 2(b). Apart from the NE-442, which contains the highest oil component, all SNEDDS formulations showed higher transmittance of over 80%. In particular, the NE-271, which has the highest amount of tween 80 in the SNEDDS system, exhibited the maximum transmittance, indicating an excellent emulsification ability.

Subsequently, the solubility of sorafenib in the selected formulations was examined. All formulations were dropped into distilled water and gently stirred for 30 min. Unloaded drugs were removed by filtering the solution through a 0.45 μm polyvinylidene fluoride membrane filter. The droplet size and PDI value were unchanged even after sorafenib encapsulation (Figure 2(c)).

The SLS-271 formulation, which had the highest amount of tween 80 in the system, showed the maximum drug solubilizing ability (Loading content: 6.6 wt %) (Figure 2(d)). At first glance, the above result is unexpected as the solubility of the drug in oil and co-solvent is much higher than that in tween 80. However, it should be considered that the hydrophilic-lipophilic balance (HLB) value in SNEDDS plays a very important role in formulation stability. The above result can be explained by the increase in the HLB value of the emulsion by tween 80, thereby increasing the emulsification ability of the formulation, and thus further improving the solubilization capacity.

The stability of sorafenib-loaded SNEDDS formulations was investigated by evaluating the particle size and PDI value for 7 days. As presented in Figure 2(e), there were no significant changes in particle size and PDI value for all formulations.

3.3. In vitro cytotoxicity of sorafenib-loaded SNEDDS formulations

Sorafenib—a multiple kinase inhibitor—has been reported to exert anticancer activity against several tumor models [4, 5, 42]. In the present study, to confirm the biological activity of the sorafenib-loaded SNEDDS formulation, the viability of the liver cancer cell line (Hep3B) and oral cancer cells (KB) after sample treatment was investigated. In the case of vehicle SNEDDS without sorafenib, no significant toxicity was observed in all cell lines up to a vehicle concentration of 100 μg/mL. Conversely, the formulation containing sorafenib showed low IC50 values in each cell line (8.07 μg/mL for Hep3B cells and 15.2 μg/mL for KB cells) (Figure 3(a)).

Figure 3.

In vitro characterization of sorafenib-loaded liquid SNEDDS. (a) Cell viability of Hep3B and KB cell populations treated with raw sorafenib, vehicle, and SLS-271. (b) Caspase-3/7 activation in Hep3B and KB cells after vehicle and SLS-271 treatment.

Subsequently, the apoptosis marker caspase 3/7 activity was evaluated 3 h or 24 h after the sample and vehicle treatment. In the sample treatment group, caspase 3/7 was remarkably activated compared to the vehicle and control, indicating that the apoptosis level was significantly increased (Figure 3(b)). These results indicate that the biological activity is preserved while the solubility and stability of sorafenib are improved in the sorafenib-loaded SNEDDS formulation.

3.4. Preparation and characterization of sorafenib-loaded solid SNEDDS

Although the liquid SNEDDS formulations have the advantage of being simple to manufacture, their disadvantages in clinical translation include leaching and drug precipitation from the capsule shell [23–25]. To overcome these issues, the liquid SNEDDS was converted into a solid SNEDDS using the spray drying method. For solid carriers, Aerosil^® 200 and PVP K30 were used as an adsorbent and a precipitation inhibitor, respectively. Aerosil^® 200, which provides a large surface area for the adsorption of SNEDDS and the PVP K30, can increase the solubilizing capacity of SNEDDS after dilution in gastrointestinal fluid [43–47].

Solid SNEDDS composed of SLS-271 and different ratios of solid carriers were prepared (Figure 4(a)), and characterization studies of each formulation including XRD, DSC, and morphology were performed. In general, when a drug is changed from a highly stabilized crystalline state to a high-energy amorphous state through the application of formulation technology, the solubility, and dissolution rate of the drug can be increased, thereby improving its bioavailability [16, 48–50]. As expected, the XRD peaks of sorafenib revealed a highly crystalline structure with diverse distinctive peaks. However, the F0, F13, F22, and F31 solid SNEDDS formulations did not have any characteristic peaks by the crystalline state of sorafenib indicating that the drug was molecularly dissolved within the SNEDDS or in an amorphous state (Figure 4(b)). The thermogram of sorafenib in Figure 4(c) shows a big sharp exothermal peak at 239 °C, due to its decomposition at its melting temperature, demonstrating crystalline characteristics. Similarly, the disappearance of drug features in solid SNEDDS formulations indicated the formation of an amorphous solid solution as the drug was molecularly dispersed in the SNEDDS system [51, 52].

Figure 4.

Preparation and characterization of sorafenib-loaded solid-SNEDDS. (a) Composition of solid SNEDDSs. Characterization of sorafenib-loaded solid-SNEDDSs. (b) XRD patterns, (c) DSC images, and (d) FE-SEM images of raw sorafenib or sorafenib-loaded solid-SNEDDS.

FE-SEM images confirmed the surface morphology of raw sorafenib, F0, F13, F22, and F31 solid SNEDDS formulations (Figure 4(d)). Raw sorafenib displayed a rectangular-shaped crystalline structure. Conversely, the sorafenib-loaded solid SNEDDS system exhibited significant changes in particle shape and surface topography. The crystalline structure of sorafenib disappeared in the F0, F13, F22, and F31 SNEDDS formulations. Furthermore, surface roughness and porosity were also enhanced in those solid SNEDDS formulations. These results indicate that sorafenib was successfully encapsulated in the SNEDDS and SNEDDS formulations were well adsorbed or coated inside the pores of solid carriers.

Subsequently, storage stability according to each temperature condition was tested with the developed SNEDDS formulations (Figure 5). The production process and storage conditions become simpler when the formulation is developed in the form of solid-SNEDDS. Therefore, it has advantages over the existing liquid form of SNEDDS formulations such as improving product stability and reducing production costs. After diversifying the storage conditions to 4 °C, 25 °C, and 37 °C, the changes in the drug content in the formulation were investigated for 4 weeks. In the case of liquid SNEDDS, the drug precipitates over time, and the initial concentration can be seen to decrease to 80% at 4 °C, 70% at 25 °C, and 60% at 37 °C (Figure 5(a)). However, in the case of solid SNEDDS, the concentration was kept constant regardless of the temperature change for 4 weeks (Figure 5(b)). These results suggest that the storage stability and economic feasibility can be further increased when sorafenib is formulated as a solid SNEDDS.

Figure 5.

Storage stability of formulations according to temperature conditions. Concentration changes of (a) sorafenib-loaded liquid SNEDDS, and (b) sorafenib-loaded solid SNEDDS over time at 4 °C, 25 °C, and 37 °C storage conditions. (c) Statistical comparison of data for remaining drug contents at different conditions. *p < 0.05, **p < 0.01.

3.5. In vitro dissolution assay

Subsequently, the dissolution profile of samples in simulated gastric and intestinal fluids was investigated according to the USP 29 apparatus 2 method. First, the dissolution profile of raw sorafenib and liquid SNEDDS formulations in simulated gastric or intestinal fluid conditions were investigated (Figure 6(a)). Under the simulated gastric and intestinal fluid conditions, raw sorafenib was barely detected due to its low solubility, but when the drugs were formulated into liquid SNEDDS, the dissolution rate of sorafenib was remarkably improved. Here, the released amount of drug in the simulated gastric condition was higher than that in the simulated intestinal fluid conditions, for both the raw drug and SNEDDS formulation. Since sorafenib is a weak basic compound, its solubility under acidic conditions could increase, which could affect the release profile. The dissolution pattern of the solid carrier and raw drug mixture prepared through the spray-drying method was also evaluated (figure S2). In this case, the solid carrier partly served as a matrix of the solid dispersion, so the dissolution rate of the drug increased slightly compared to the raw drug, but there was no remarkable improvement.

Figure 6.

The dissolution profiles of (a) raw sorafenib and sorafenib-loaded liquid SNEDDS (SLS-271), and (b) sorafenib-loaded solid SNEDDS in simulated gastric (pH 1.2) or intestinal (pH 6.8) conditions.

Finally, the developed solid SNEDDS formulations were used to determine the dissolution pattern of the drug in gastric and intestinal fluid conditions (Figure 6(b)). Among the solid SNEDDS, F0 using only Aerosil^® 200 as a solid carrier showed an approximately 35% dissolution amount of sorafenib within 5 min, which was maintained up until 120 min in simulated gastric conditions. However, when the PVP K30 polymer—a recrystallization inhibitor [45, 47]—was added to solid SNEDDS, the dissolution of sorafenib was significantly increased. The released amount of drug from F13, F22, and F31 reached a plateau level within 30 min, showing a total amount of about 95% for F13, F22, and F31. The dissolution profile of F13, F22, and F31 in simulated intestinal fluid conditions was similar to those in the simulated gastric conditions.

3.6. In vitro transport assay in Caco-2 cells

Subsequently, the level of in vitro penetration of raw sorafenib and sorafenib-loaded solid SNEDDS (F22) was investigated. Samples containing the equivalent amount of sorafenib were dispersed in cell culture media, stirred for 5 min, and treated to a trans-well in which the Caco-2 cell monolayer was placed. By analyzing the amount of drug penetration by time, it was confirmed that F22 began to be detected at 30 min of treatment, and steadily increased during the incubation time of 240 min. However, in the case of the raw drug, there was absolutely no penetration due to its low solubility. The TEER values of cell monolayers were not changed after the experiment, indicating that the integrity of the cell monolayers was maintained during the entire time period of the permeation study of sorafenib from SNEDDS formulations.

3.7. In vivo pharmacokinetic study

In this study, the pharmacokinetic profile of sorafenib-loaded S-SNEDDS formulation (F22) was compared to that of free sorafenib in rats. The graph in Figure 8(a) shows the mean plasma concentration of sorafenib versus time for both the F22 and free sorafenib when given orally at a dose of 50 mg/kg of sorafenib. The pharmacokinetic parameters (C _max, T _max, AUC, K _el, t _1/2) are summarized in Figure 8(b). The results showed that the F22 formulation resulted in significantly higher plasma concentrations compared to the free drug (p < 0.05). Specifically, the C _max (6.1 ± 0.6 μg/mL) and AUC (136.1 ± 25.9 h×μg/mL) values of the F22 treated group were 2.8-fold and 4.6-fold higher than those of the free drug treated group, respectively. The observed increase in the oral bioavailability of sorafenib in the S-SNEDDS could be attributed to a noticeable improvement in the dissolution rate and subsequent absorption of the drug in rats.

Figure 8.

Plasma concentration-time profiles of sorafenib in rats after oral administration (n = 3). Non-compartmental analysis was used to calculate various pharmacokinetic parameters. C_max (Maximum plasma concentration); T _max (Time required to reach C _max); AUC (Area under the plasma concentration versus time curve from zero to 48 hours); K _el (Elimination rate); T_1/2 (Half-life).

4. Discussion

Sorafenib has been commercialized as a tyrosine kinase inhibitor for the treatment of several cancers. However, the current formulation of sorafenib tablet (Nexavar^®) shows a low bioavailability of about 30–50 %, thus it is desirable to improve the bioavailability through additional formulation studies [53].

Song and Truong et al. developed a sorafenib-loaded solid dispersion and demonstrated that a successfully improved bioavailability by releasing an amorphous drug from the matrix [54, 55]. However, it is often reported that solid dispersion formulations have lower drug solubility or bioavailability than SNEDDS formulations. Several studies have confirmed that the dissolution rate and bioavailability could be improved when a specific modified technique is applied to a solid dispersion preparation [49, 56]; however, considering that the dissolution and bioavailability may vary depending on the target drug, it is necessary to diversity the formulation technology.

Recently, there has been active progress in the research of enhancing the solubility and bioavailability of sorafenib using the SNEDDS technology [32, 33]. However, although SNEDDS have several benefits, including increased drug solubility, enhanced bioavailability, and reduced toxicity, they also have some drawbacks, with the most notable being storage instability. Storage stability is a critical issue with SNEDDS. Since SNEDDS are composed of a mixture of oils, surfactants, and co-surfactants, they can be prone to physical and chemical instability over time. The oil and surfactant can separate, leading to phase separation, creaming, or coalescence, which can affect the uniformity of the drug dose and its absorption. The storage stability of SNEDDS can be improved by developing solid-SNEDDS (S-SNEDDS). S-SNEDDS are essentially SNEDDS that have been converted into solid dosage forms such as powders, pellets, or tablets. The conversion of SNEDDS into solid forms can help to stabilize the formulation by preventing the separation of the oil and surfactant. S-SNEDDS also have improved physical stability, reduced sensitivity to environmental factors, and increased shelf-life compared to liquid SNEDDS. As depicted in Figure 5, solid SNEDDS exhibit significantly improved storage stability under various conditions compared to conventional liquid SNEDDS. While previous studies have utilized liquid SNEDDS for the formulation of sorafenib, our research employs an advanced solid-SNEDDS formulation that addresses the stability issues associated with traditional SNEDDS, offering a more advantageous formulation form for commercialization. Since it is easier to store and maintain stability in the solid dosage form of the drug compared to the liquid dosage form, a solid dosage form is preferable if there is no significant difference in the dissolution pattern [23, 24, 57].

Toxicity was considered in selecting the composition of the SNEDDS formulation. Therefore, non-ionic surfactants with relatively low toxicity were screened, and tween 80, which is currently FDA-approved and most used, was selected. Labrasol was also one of the candidates; however, it was excluded as the HLB value, which could affect the emulsification ability of SNEDDS, was lower than that of tween 80 [38, 39]. It has been reported that in the development of the above SNEDDS formulation, tween 80 used can inhibit the p-glycoprotein function in oral adsorption, so the above compositions may help the absorption of the drug [58–60].

It is well known that solidification of SNEDDS formulations can be successfully achieved using Aerosil^® 200. Recently, there have been reports of the possibility of PVP not only being used as a solid dispersion matrix for some drugs, but also for inhibiting the precipitation of a drug released from the formulation when PVP is mixed with Aerosil^® 200 [46, 61], thereby increasing the stability and dissolution rate of the overall SNEDDS formulation.

In the present study, optimization of solid SNEDDS was conducted through an investigation of the appropriate combination of Aerosil^® 200 and PVP. Although the various combination ratios of Aerosil^® 200 and PVP did not significantly affect the drug dissolution rate, it was clearly confirmed that the addition of PVP had a remarkable effect on the drug dissolution rate. Unlike the solid SNEDDS, the dissolution rate of the drug from liquid SNEDDS was limited to 80% at pH 1.2 and 60% at pH 6.8. This could be a phenomenon caused by the precipitation of some drugs after being released from the emulsion. Sorafenib is a moderately basic compound, thus its solubility in the intestinal condition is very low. Therefore, the total dissolution amount from raw sorafenib and F0 was lower than that in simulated gastric conditions.

In S-SNEDDS, the drug is dissolved or dispersed in a lipid-based matrix that is then solidified to form a solid dosage form. Upon administration, the solid dosage form comes into contact with the aqueous environment of the gastrointestinal tract, which leads to the formation of an oil-in-water nanoemulsion. The solubilized drug is then released by diffusion from the nanoemulsion into the aqueous environment, where it can be absorbed. Factors such as the nature of the lipid matrix, surfactant and cosurfactant composition, drug loading, and the pH of the gastrointestinal tract can all influence the drug release kinetics from S-SNEDDS [62, 63].

Our study showed that the stability of the formulation can be significantly improved when using solid SNEDDS compared to liquid SNEDDS. Since stability varies depending on storage conditions in the case of liquid SNEDDS, additional costs are required to maintain quality during the commercialization process. Therefore, solid SNEDDS with excellent storage stability can be preferred for drug development.

In conclusion, our results highlight the use of solid SNEDDS formulation as an alternative to the conventional sorafenib formulation to improve the bioavailability and outline potential translational directions for oral drug development.

5. Conclusion

In this study, we developed a solid-SNEDDS formulation containing sorafenib, a multiple tyrosine kinase inhibitor. This formulation not only improved drug solubility but also dramatically enhanced the storage stability, which is a major limitation of the conventional liquid form of SNEDDS. Compared to free drug, the developed formulation showed improved drug release and permeability in vitro, and increased bioavailability in vivo. Overall, the potential benefits of this formulation make it a promising candidate for clinical translation in the treatment of cancer. With further investigation and regulatory approval, this drug delivery system has the potential to offer a significant advancement in cancer treatment.

Figure 7.

Permeation amount of sorafenib across Caco-2 cells monolayer treated with raw sorafenib and sorafenib-loaded solid SNEDDS (F22). Each value represents means ± standard deviation (n = 3).