Formulation of Nanomicelles to Improve the Solubility and the Oral Absorption of Silymarin

Abstract

Two novel nanomicellar formulations were developed to improve the poor aqueous solubility and the oral absorption of silymarin. Polymeric nanomicelles made of Soluplus and mixed nanomicelles combining Soluplus with d-α-tocopherol polyethylene glycol 1000 succinate (vitamin E TPGS) were prepared using the thin film method. Physicochemical parameters were investigated, in particular the average diameter, the homogeneity (expressed as polydispersity index), the zeta potential, the morphology, the encapsulation efficiency, the drug loading, the critical micellar concentration and the cloud point. The sizes of ~60 nm, the narrow size distribution (polydispersity index ≤0.1) and the encapsulation efficiency >92% indicated the high affinity between silymarin and the core of the nanomicelles. Solubility studies demonstrated that the solubility of silymarin increased by ~6-fold when loaded into nanomicelles. Furthermore, the physical and chemical parameters of SLM-loaded formulations stored at room temperature and in refrigerated conditions (4 °C) were monitored over three months. In vitro stability and release studies in media miming the physiological conditions were also performed. In addition, both formulations did not alter the antioxidant properties of silymarin as evidenced by the 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay. The potential of the nanomicelles to increase the intestinal absorption of silymarin was firstly investigated by the parallel artificial membrane permeability assay. Subsequently, transport studies employing Caco-2 cell line demonstrated that mixed nanomicelles statistically enhanced the permeability of silymarin compared to polymeric nanomicelles and unformulated extract. Finally, the uptake studies indicated that both nanomicellar formulations entered into Caco-2 cells via energy-dependent mechanisms.

Keywords: Caco-2 cell line; PAMPA; antioxidant activity; drug delivery; mixed nanomicelles; oral bioavailability; polymeric nanomicelles; silymarin.

Figure 1

Visual appearance of the nanomicelles. (A) Empty polymeric nanomicelles (PNM); (B) Empty mixed nanomicelles (MNM); (C) Silymarin (SLM)-loaded PNM; (D) SLM-loaded MNM; (E) SLM aqueous suspension.

Figure 2

Visual appearance of the fluorescent nanomicelles. (A) FITC-loaded PNM; (B) FITC-loaded MNM.

Figure 3

(A) Transmission electron microscope (TEM) image of SLM-loaded polymeric nanomicelles (SLM-PNM). Scale bar 200 nm; (B) TEM image of SLM-loaded mixed nanomicelles (SLM-MNM). Scale bar 200 nm.

Figure 4

Effective permeability (P_e) of free-silymarin (Free-SLM), silymarin-loaded polymeric nanomicelles (SLM-PNM) and silymarin-loaded mixed nanomicelles (SLM-MNM). Data are expressed as mean ± SD, n = 3. ** p < 0.01 vs. free silymarin, by Kruskal-Wallis test and Dunn’s multiple comparisons test.

Figure 5

Representative analysis of P-gp expression in Caco-2 cells. PCR products were separated on 1.8% agarose gel containing Safeview.

Figure 6

Cellular uptake of FITC-PNM (A) and FITC-MNM (B), green staining, in Caco-2 cells after 1 h of exposure at 37 °C (100%), at 4 °C or in the presence of endocytic inhibitors. Nuclei stained with DAPI. Final magnification 20×, scale bar 50 µm. Results are expressed as mean ± SEM, n = 3. ** p < 0.01 vs. 37 °C; * p < 0.05 vs. 37 °C, by Kruskal-Wallis test and Dunn’s multiple comparisons test.

Figure 7

Antioxidant activity of SLM in solution, SLM-loaded polymeric nanomicelles (SLM-PNM), SLM-loaded mixed nanomicelles (SLM-MNM), empty polymeric nanomicelles (PNM) and empty mixed nanomicelles (MNM) (Mean ± SD, n = 3).