Nanoemulsion formulation boosts flutamide solubility and kills prostate cancer cells in lab tests

Flutamide, a nonsteroidal antiandrogen used to treat prostate cancer, dissolves so poorly in water that its oral bioavailability remains a persistent challenge. A research team has now formulated and optimized a self-nanoemulsifying drug delivery system, or SNEDDS, for the drug, using sesame oil as the oil phase, Tween 20 as the surfactant, and PEG 400 as the co-surfactant. The optimized formulation emulsified itself in 7.9 seconds, released 98.69 percent of its drug payload in vitro, and formed nanoscale droplets averaging 120.1 nanometers in diameter. In cytotoxicity testing on PC-3 prostate cancer cells, the formulation achieved an IC50 of 177.7 micrograms per milliliter, substantially lower than the 644.8 micrograms per milliliter measured for the pure drug, indicating markedly stronger cell-killing activity at lower concentrations.

The formulation strategy exploits the way SNEDDS behave once they enter the gastrointestinal tract. These isotropic blends of oil, surfactant, and co-surfactant spontaneously generate an oil-in-water emulsion when they contact aqueous fluids, dispersing into droplets small enough to vastly expand the surface area available for drug absorption and digestion. Because the emulsification is driven by gentle peristaltic motion rather than external energy, the approach suits drugs classified as BCS Class II or IV, whose poor solubility limits how much of an oral dose actually reaches the bloodstream. The researchers also incorporated rutin, a flavonoid, into the system alongside flutamide, reasoning that flavonoids can modulate reactive oxygen species, induce cell cycle arrest, and trigger apoptosis in cancer cells while behaving as antioxidants in normal tissue, potentially reducing the side effects associated with flutamide therapy.

Selecting the right excipients required systematic solubility screening. Flutamide was tested in sesame, olive, sunflower, and soybean oils, and sesame oil dissolved the drug most effectively, with a solubility value near 0.8 compared with roughly 0.6 for olive oil and about 0.4 for both sunflower and soybean oils. Among surfactants, Tween 80 showed the highest solubility at approximately 0.8, with Tween 20 close behind, while Span 80 and Span 20 lagged at around 0.4, a difference the authors attribute to the lower hydrophilic-lipophilic balance values of the Span series. In the co-surfactant screen, PEG 400 outperformed propylene glycol and PEG 1000, approaching a solubility value of 0.8, which led to its selection alongside Tween 20 at a 2:1 surfactant-to-co-surfactant ratio.

That ratio was confirmed through a pseudo-ternary phase diagram built by aqueous titration. Surfactant and co-surfactant were mixed in 1:1, 1:2, and 2:1 proportions, then combined with oil across ratios ranging from 1:9 to 9:1 and titrated with water to map where stable emulsions form. The 2:1 Smix ratio produced the largest self-emulsifying region, indicating that this proportion most effectively lowers interfacial tension and enables spontaneous droplet formation upon water contact. The authors note that higher Smix content broadens the emulsification range through enhanced solubilization capacity, though excessive surfactant concentration raises toxicity concerns, making the shaded region of the diagram the practical target zone for formulation development.

Optimization proceeded through a Box-Behnken design, a response surface methodology that evaluates three independent variables, namely the concentrations of oil, surfactant, and co-surfactant, at three levels each while minimizing the number of experimental runs. Two dependent variables guided the search: self-emulsification time and percent cumulative drug release. Analysis of variance was applied to both responses, and three-dimensional and contour plots generated in Design Expert software revealed how each factor influenced the outcomes. An overlay plot superimposed the response surfaces to identify the ideal formulation, and the predicted values were compared against observed results to calculate prediction error. The optimized batch emerged from this statistical exercise with the rapid 7.9-second emulsification and the 98.69 percent cumulative release that define its performance profile.

Particle characterization by dynamic light scattering confirmed the nanoscale architecture. The Z-average diameter came in at 120.1 nanometers with a polydispersity index of 0.384, a value the authors describe as indicating moderate size uniformity within acceptable limits for nanoformulations. The primary intensity peak sat at 167.3 nanometers and accounted for 96.9 percent of scattering intensity, while a minor secondary peak at 4685 nanometers contributed just 3.1 percent, likely representing a small population of aggregated particles. Zeta potential measured −0.0179 millivolts with a standard deviation of 3.18 millivolts, a value close to zero that suggests the formulation relies on steric stabilization rather than electrostatic repulsion, consistent with the non-ionic surfactant in the system. The authors note that higher absolute zeta potentials, typically above ±30 millivolts, generally signal stronger electrostatic stabilization in colloidal systems.

Transmission electron microscopy provided direct visual confirmation of nanoscale morphology, with images captured at a resolution scale of 100 nanometers showing well-defined structural features and homogeneous dispersion. Differential scanning calorimetry compared the thermal behavior of pure flutamide with the optimized SNEDDS. The pure drug displayed two endothermic transitions, one peaking at 80.59 degrees Celsius with an enthalpy of 70.915 joules per gram and another at 109.62 degrees Celsius with an enthalpy of 72.256 joules per gram, suggesting distinct crystalline or polymorphic forms. The optimized formulation showed a single endothermic transition peaking at 83.70 degrees Celsius with a lower enthalpy of 52.011 joules per gram, a pattern the authors interpret as reflecting reduced crystallinity or molecular dispersion of the drug within the formulation, a state known to enhance solubility and dissolution rates.

Stress testing reinforced the physical robustness of the system. The formulation withstood six heating and cooling cycles between 40 and 4 degrees Celsius without phase separation, precipitation, or turbidity, and remained intact through a centrifugation challenge with no signs of creaming or cracking. Accelerated stability testing under International Council for Harmonisation guidelines for climatic zone IV, conducted at 40 degrees Celsius and 75 percent relative humidity over six months, showed no significant changes in drug content, zeta potential, particle size, or physical appearance. Fourier-transform infrared spectroscopy further confirmed chemical compatibility, as the absorption bands of the optimized formulation matched those of pure flutamide, with characteristic peaks including C–H stretching near 2921.71 and 2850.82 inverse centimeters and C=O stretching at 1742.20 inverse centimeters, indicating that flutamide’s functional groups remained stable in the mixture.

The cytotoxicity results carry the most immediate therapeutic interest. In the MTT assay, PC-3 cells were seeded at 10,000 cells per well and exposed to varying concentrations of the liquid formulation. Cell viability declined progressively from 77.89 percent at the lowest tested concentration of 0.78125 microliters per milliliter to 33.42 percent at the highest concentration of 50 microliters per milliliter, demonstrating a clear dose-dependent effect. The half-maximal inhibitory concentration fell between 3.125 and 6.25 microliters per milliliter in the viability curve, and the IC50 value of 177.7 micrograms per milliliter for the optimized formulation, when compared against 644.8 micrograms per milliliter for the pure drug, underscores the potency gain achieved through nanoemulsification. The authors suggest that mechanisms such as mitochondrial dysfunction, reactive oxygen species generation, and DNA damage leading to apoptosis or cell cycle arrest may underlie the observed cytotoxicity, though they emphasize that further work is needed to evaluate effects on normal prostate cells and to establish a therapeutic index.

Drug release kinetics were fitted to zero-order, first-order, and Higuchi models, with the near-complete release profile showing a strong correlation coefficient of 0.9883 and a slope consistent with steady, controlled delivery. The authors frame the work as a promising strategy for enhancing bioavailability and therapeutic effectiveness in prostate cancer treatment, but they are explicit that the findings remain at the in vitro stage. Future studies, they write, should focus on in vivo pharmacokinetics and safety profiles to confirm clinical applicability, including toxicological evaluations to address long-term safety and clinical trials to demonstrate effectiveness and benefits for patient compliance. The scalability and adaptability of the SNEDDS platform, they add, could extend to other poorly soluble medications beyond flutamide, opening broader applications in cancer treatment and adjacent therapeutic areas.

Subject of Research: Development of a self-nanoemulsifying drug delivery system to improve the solubility, release, and anticancer activity of flutamide

Article Title: Formulation and optimization of self-nanoemulsifying drug delivery systems (SNEDDS) for enhancing the solubility, bioavailability, and anticancer activity of flutamide

Article References: Sapra, K., Sharma, R., Samaddar, S., & Sardana, S. (2026). Formulation and optimization of self-nanoemulsifying drug delivery systems (SNEDDS) for enhancing the solubility, bioavailability, and anticancer activity of flutamide. Discover Pharmaceutical Sciences, 2(1), Article 4. https://doi.org/10.1007/s44395-026-00037-5

Image Credits: AI Generated

DOI: 10.1007/s44395-026-00037-5

Keywords: flutamide, SNEDDS, prostate cancer, drug solubility, nanoemulsion, PC-3 cells, Box-Behnken design, in vitro drug release, cytotoxicity, rutin, sesame oil, zeta potential