Optimization of a Luteolin-Loaded TPGS/Poloxamer 407 Nanomicelle: The Effects of Copolymers, Hydration Temperature and Duration, and Freezing Temperature on Encapsulation Efficiency, Particle Size, and Solubility

Abstract

Background: Luteolin is a flavonoid compound that has been widely studied for its various anti-cancer properties and sensitization to multidrug-resistant cells. However, the limited solubility and bioavailability of Lut hindered its potential clinical use. Theoretically, the combination of this compound with vitamin E TPGS and poloxamer 407 can produce a synergistic effect to enhance tumor apoptosis and P-glycoprotein inhibition. This study aimed to develop and optimize vitamin E TPGS/Poloxamer 407 micelles loaded with luteolin through investigating certain factors that can affect the encapsulation efficiency and particle size of the micelle.

Methods: A micelle was prepared using the film hydration method, and the micellar solution was lyophilized. The cake formed was analyzed. The factors investigated include the concentrations of the surfactants, ratio of vitamin E TPGS/Poloxamer 407, temperature of the hydrating solution, duration of hydration, and freezing temperature before lyophilization. The effects of these factors on the encapsulation efficiency and particle size of the micelle were also studied. The encapsulation efficiency was measured using a UV-Vis spectrophotometer, while particle size was measured using dynamic light scattering.

Results: The optimized micelle was found to have 90% encapsulation efficiency with a particle size of less than 40 nm, which was achieved using a 10% concentration of surfactants at a vitamin E TPGS/Poloxamer 407 ratio of 3:1. The optimized temperature for hydrating the micellar film was 40 °C, the optimized mixing time was 1 h, and the optimized freezing temperature was -80 °C. The solubility of the luteolin-loaded micelles increased 459-fold compared to pure Lut in water. The critical micelle concentration of the vitamin E TPGS/Poloxamer 407 micelle was 0.001 mg/mL, and the release study showed that luteolin-loaded micelles exhibited sustained release behavior. The release of luteolin from a micelle was found to be higher in pH 6.8 compared to pH 7.4, which signified that luteolin could be accumulated more in a tumor microenvironment compared to blood.

Conclusion: This study demonstrated that several factors need to be considered when developing such nanoparticles in order to obtain a well-optimized micelle.

Keywords: film hydration method; freeze-drying temperature; hydration temperature; luteolin; micelle.

Figure 1

PS of the optimized micelle obtained via (A) dynamic light scattering (DLS) and (B) transmission electron microscope (TEM).

Figure 2

Critical micelle concentration (CMC) of the optimized micelle determined via KI/I₂ probe. Blue dot is the absorbance at the given concentration. Black lines are linear curves of ‘gradual increase’ (from −6 to −3) in absorbance and ‘rapid increase’ (from −3.3 to −1.6) in absorbance. Black arrow determines the CMC where the two black lines intersected.

Figure 3

Fourier-transform infrared (FTIR) spectrum of (A) Lut, (B) TPGS, (C) Pol, and (D) sample.

Figure 3

Fourier-transform infrared (FTIR) spectrum of (A) Lut, (B) TPGS, (C) Pol, and (D) sample.

Figure 4

Crystallinity analysis of (A) Lut, (B) Pol, (C) TPGS, (D) blank micelle and (E) Lut-loaded micelle via XRD.

Figure 4

Crystallinity analysis of (A) Lut, (B) Pol, (C) TPGS, (D) blank micelle and (E) Lut-loaded micelle via XRD.

Figure 4

Crystallinity analysis of (A) Lut, (B) Pol, (C) TPGS, (D) blank micelle and (E) Lut-loaded micelle via XRD.

Figure 5

Drug release study of free Lut (grey), Lut-loaded micelle in pH 7.4 (blue), and Lut-loaded micelle in pH 6.8 (orange).