The dissolution medium was 900 mL of distilled water and the temperature was maintained at 37 ± 0.5 °C.
The speed of the paddle was set to 50 rpm.
| Ingredient | Concentration | Function | Notes |
|---|---|---|---|
| Sildenafil citrate | 10 mg/mL | Active pharmaceutical ingredient | Main therapeutic agent |
| Purified water | - | Solvent | Ensures proper dissolution |
| Preservative (e.g., parabens) | 0.1% | Prevent microbial growth | Preserves solution stability |
| Flavouring agents | - | Improve palatability | Fruit or mint flavors |
| Sweeteners | - | Mask bitterness | Aspartame or sucralose |
At specific time intervals (0, 5, 10, 15, 30 and 45 min), an aliquot (3 mL) of the sample was collected, filtered through a 0.45 µm nylon syringe filter and assayed for the content of sildenafil by employing HPLC, as described above.
To render the sample electrically conductive, a platinum coating (4 min at 15 mA) was applied by utilising the EmiTeck Sputter Coater (K575 K) at a speed of 6 nm/min under vacuum (0.8 Pa). Particle-size analysis was carried out using Mastersizer 3000 (Malvern, Worcestershire, UK). The instrument was optimised under the following conditions: air pressure, 1 bar; feed rate, 50%; gab, 1. The thermal analysis was conducted via a differential scanning calorimetry (DSC Q200; TA Instruments, New Castle, DE, USA). Each sample (approximately 5 mg) was placed in standard aluminium pans and dry nitrogen was employed as the effluent gas.
All samples were scanned at a temperature ramp speed of 10 °C/min and heat flow from 0 to 300 °C. X-ray powder scattering measurements were performed by means of an X-ray diffractometer (D/MAX-2500, Rigaku, Japan) at room temperature. An angular increment of 0.02° per second was selected over a range of 2θ angles from 3° to 50°. Dissolution profiles of sildenafil-loaded amorphous microspheres, crystalline microspheres, solid SNEDDS and the drug powder (equivalent to 10 mg of sildenafil) were investigated using Vision Classic 6 (Hanson Research Co.; Los Angeles, CA, USA). USP apparatus II (paddle method) was utilised. Twenty-four rats were non-selectively divided into four groups (each group consisted of six rats).
After the anaesthesia process, the femoral artery of the rats was inserted with a surgical tube and fixed on a surgical board.
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Before surgery, heparin (50 IU/mL) was prepared to coat the inside of the polyethylene tube.
| Drug Class | Potential Effect | Clinical Note |
|---|---|---|
| Nitrates | Severe hypotension | Absolute contraindication |
| Alpha-blockers | Increased risk of hypotension | Use with caution |
| CYP3A4 inhibitors (e.g., ketoconazole) | Increased sildenafil levels | Dose adjustment needed |
| Other vasodilators | Enhanced vasodilatory effects | Monitor blood pressure |
The sildenafil-loaded amorphous microspheres, crystalline microspheres, solid SNEDDS and the drug powder were placed in a #9 gelatin capsule and administered orally at a dose of 20 mg/kg.
Additionally, the nanoemulsion droplet size was assessed only for the formulations providing excellent nanoemulsifying ability (achieving the visual standard of the preceding experiment). The nanoemulsion droplet size was analysed via a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Among the formulations, the liquid SNEDDS with the smallest emulsion droplet size was selected. Next, sildenafil was incorporated into the chosen liquid SNEDDS and highly porous inorganic materials with large specific surface areas (500 mg) were added for solidification. The suspension flowed through a pneumatic nozzle of 0.7 mm diameter in a Büchi mini spray dryer (B-290; Flawil, Switzerland) at a flow rate of 5.0 mL/min.
The inlet temperature was set at 130 °C and the outlet temperature was maintained at 80 °C. The aspirator was set at 90% and the pressure of the spraying air was 4 kg/cm2. During the entire spray-drying process, the suspension was continuously stirred (500 rpm) using a magnetic bar to maintain a stable suspension state. The shape and surface of the samples were examined by employing an S-4800 scanning electron microscope (Hitachi; Tokyo, Japan). Double-sided adhesive tape was utilized to fix the samples. Plasma samples (300 µL) were collected at predetermined time points via the cannulated tube.
After centrifuging the sample at 20,000 g for 10 min, the supernatant (100 µL) was separated. The protein precipitation method was applied while using revaprazan as an internal standard. Finally, the amount of sildenafil was analysed through HPLC.
Approximately 100 mg of sildenafil (to reach a supersaturated state) was added to 1 mL of 1% (w/v) hydrophilic polymer aqueous solution (PVP, Na-CMC, gelatin, PVA, HP-β-CD, HPC-L HPMC and dextran), 5% (w/v) surfactant aqueous solution (Tween 80, Span 80, Cremophor EL, Labrafil M2125CS, Solutol HS15, Plurol diisosterarique Capryol 90, Lauroglycol FCC, poloxamer 188, PEG 4000, Transcutol HP and Labrasol), and pure oil (mineral oil, soybean oil, corn oil, olive oil, linseed oil and Captex 300) to perform the solubility test. After 5 min of vortex-mixing, the samples were placed in a shaking water bath (Daihan Scientific, Wonju, South Korea) for 5 days (25 °C and shaking speed of 100 rpm) to generate a supersaturated state. Three days later, the samples were centrifuged (10,000 × g for 15 min) using a 5430R centrifuge (Eppendorf, Hamburg, Germany). Next, the supernatant was filtered (0.45 μm, nylon) and diluted with the mobile phase for analysing sildenafil by utilising a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, CA, USA). The HPLC system was arrayed with the Agilent Chem Station software, G1311C 1260 Quat Pump and G1314B 1260 VWD VL detector.
The mobile phase, column and gradient methods used were consistent with those used in previous studies.20 According to the aqueous solubility tests, suitable polymer and surfactant were chosen. Sildenafil-loaded amorphous microspheres were manufactured by mixing numerous ratios of polymer, surfactant and sildenafil in ethanol. The experimental plan of formulations is shown in Table S1. The resultant clear solution was introduced into a Büchi mini spray dryer and continuously pushed to the pneumatic nozzle (0.7 mm diameter) by employing a peristaltic pump at a flow rate of 5.4 mL/min. The inlet temperature was set at 90 °C and the outlet temperature was maintained at 60–70 °C. Statistically significant difference was confirmed by the Student’s t-test (for a pair of groups) and one-way ANOVA followed by Tukey’s post hoc test (for more than two groups).
Spray-dryer was used to fabricate each drug delivery system. Spray-drying was chosen due to its widespread use in pharmaceutical industry and laboratory-scale experiments.25–27 Compared to other techniques, such as supercritical fluid-based approach or microfluidics, spray-dryer has several advantages including continuous mode of operation, reproducibility and low production cost.28–30 Amorphous microspheres, crystalline microspheres and solid SNEDDS were compared according to aqueous solubility, dissolution profile and physicochemical properties. The schematic design of this study is illustrated in Figure 1. Each physicochemical property was expected to induce different solubilising mechanisms and aqueous microenvironments.31 These differences resulted in performance order in aqueous solubility, according to the previous studies.32 Moreover, pharmacokinetic studies in rats were conducted to compare the oral bioavailability of sildenafil among amorphous microspheres, crystalline microspheres, solid SNEDDS and drug powder. Finally, a drug delivery system exhibiting the highest oral bioavailability of sildenafil was selected.
Sildenafil (base) was supplied by Huons Pharm. Polyvinylpyrrolidone (PVP, K30), Solutol HS15 and Cremophor EL were purchased from BASF (Ludwigshafen, Germany). Dextran, polyvinyl alcohol (PVA), hydroxypropyl methylcellulose 2910 (HPMC), pyrogenic silica (HDK N20 Pharma), hydroxypropyl cellulose low viscosity (HPC-L), carboxymethyl cellulose sodium (Na-CMC), poloxamer 188, PEG 4000 and hydroxypropyl beta-cyclodextrin (HP-β-CD) were kindly offered from Hanmi Pharm. Soybean oil, sorbitan monooleate (Span 80), olive oil, polysorbate (Tween 80) and corn oil were provided by Daejung Chem. Medium-chain triglycerides (Captex 300) were purchased from Abitec (Columbus, OH, USA). For the statistical calculation, SPSS Version 26 (IBM, Armonk, NY, USA) was utilised.
The aspirator was fixed at 100% and the pressure of the spraying air was 4 kg/cm2. To optimise the ratio of polymer, surfactant and sildenafil, solubility and dissolution tests were performed. After selecting the configuration of amorphous microspheres, the solvent was changed from ethanol to distilled water to manufacture the crystalline microspheres. Then, the resultant suspension was spray dried while continuously stirring using a magnetic bar (500 rpm). The inlet temperature was maintained at 140 °C and the outlet temperature was set at 80–90 °C.
The pneumatic nozzle, pump rate, aspirator value and spraying air pressure sildenafil citrate tablets 50 mg were consistent with the preparation conditions of the amorphous microspheres. The composition of liquid SNEDDS was selected on the basis of an aqueous solubility test. Afterwards, numerous ratios of oil, surfactant and co-surfactant were combined to create liquid SNEDDS formulations. The formulations containing various mixtures of oil, surfactant and co-surfactant were plotted in ternary-phase diagram (Figure S1). Furthermore, their dispersion in water was visually evaluated. A p-value of <0.05 was considered significant.33 Among the investigated ingredients, PVP and Labrasol were selected as a proper polymer and surfactant, respectively, due to their high solubility as shown in Figure S2-A and B.
Afterwards, various amorphous microspheres were prepared (Table S1).
Mineral oil and linseed oil were obtained from Samchun Chemical Co. (Pyeongtaek, South Korea). Gelatin and sesame oil were purchased from Wako Chemicals Co. (Tokyo, Japan). All other chemicals and solvents were of reagent grade; they were used with or without further purification.
Male Sprague-Dawley (SD) rats weighing 300 ± 20 g were supplied by Orient Bio (Sungnam, South Korea). All healthy rats were stored in animal cages at 25–27 °C and 55 ± 5% relative humidity during the whole process. The SD rats were fasted for 12 h; however, they were allowed free access to water before drug administration and pharmacokinetic handling. The entire animal care and processes for the animal studies were conducted according to the NIH Policy. Additionally, the protocols of pharmacokinetic studies were accepted by the Institutional Animal Care and Use Committee (IACUC) at Hanyang University.