Development of a novel microemulsion for oral absorption enhancement of all-trans retinoic acid

Abstract

This study was aimed to develop a novel microemulsion that contained oleth-5 as a surfactant to enhance the oral absorption of all-trans retinoic acid (ATRA). The prepared microemulsion was evaluated for its particle size, shape, zeta potential, in vitro release, in vitro intestinal absorption, intestinal membrane cytotoxicity and stability. The obtained microemulsion was spherical in shape with a particle size of <200 nm and a negative surface charge. The in vitro release of the ATRA-loaded microemulsion was best fit with the zero-order model. This microemulsion significantly improved the intestinal absorption of ATRA. Confocal laser scanning microscopy analysis using a fluorescent dye-loaded microemulsion also confirmed the intestinal absorption result. The intestinal membrane cytotoxicity of the ATRA-loaded microemulsion did not differ from an edible oil (fish oil). Stability testing showed that the ATRA-loaded microemulsion was more stable at 25°C than 40°C.

Keywords: all-trans retinoic acid; fish oil; microemulsion; oleth-5; oral absorption enhancement.

Figure 1

Structure of all-trans retinoic acid.

Figure 2

Pseudo-ternary phase diagram composed of fish oil as the oil phase with various surfactant mixtures (A) oleth-5 and Transcutol P (1:1), (B) oleth-5 and Cetiol HE (1:1), (C) oleth-5 and Tween 20 (1:1) and (D) oleth-5 and Tween 80 (1:1) as surfactant and co-surfactant, respectively. The gray area represents the microemulsion region.

Figure 3

Pseudo-ternary phase diagram composed of fish oil as the oil phase with various surfactant and co-surfactant mixture (oleth-5 and Transcutol P) ratios (A) 1:1, (B) 2:1, (C) 3:1 and (D) 4:1. The gray area represents the microemulsion region.

Figure 4

Transmission electron microscopy images of (A) ATRA-loaded ME 1, (B) ATRA-loaded ME 2, (C) ATRA-loaded ME 3 and (D) ATRA-loaded ME 4. Magnification ×200. Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 5

The rheological behaviors of ATRA-loaded ME 1 (Img), ATRA-loaded ME 2 (Img), ATRA-loaded ME 3 (Img) and ATRA-loaded ME 4 (Img). All data represent the (mean ± standard deviation (n=3). Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 6

The in vitro percent cumulative release profiles of all-trans retinoic acid from the microemulsion formulations ME 1 (Img), ME 2 (Img), ME 3 (Img) and ME 4 (Img). All data represent the mean ± standard deviation (n=3). Abbreviation: ME, microemulsion.

Figure 7

Confocal laser scanning microscopy images show x–z plane serial penetration of a porcine intestine treated with (A1) nile red in fish oil and (B1) nile red-loaded ME 4 at a time of 3 h, scale bar represents 100 µm. The (A2) and (B2) images are the intensity projections through the z-axis of (A1) and (B1), respectively. The scale bar represents 100 µm. Abbreviation: ME, microemulsion.

Figure 8

Comparison of fluorescence intensity profiles of nile red at various depths into the intestine shown in Figure 7. All data represent the mean ± standard deviation. Abbreviation: AU, arbitrary unit.

Figure 9

H&E-stained porcine intestine treated with (A) phosphate-buffered saline, (B) 3% w/w Triton X-100, (C) ATRA in fish oil and (D) ATRA-loaded ME 4. Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.

Figure 10

The percentage of ATRA remaining in ME 4 after storage for 0, 1, 2, 3, 4, 5 and 6 months after preparation at 25°C (■) and 40°C (□). Each value represents the mean ± standard deviation (n=3). Abbreviations: ATRA, all-trans retinoic acid; ME, microemulsion.