Preparation, characterization, and evaluation of antitumor effect of Brucea javanica oil cationic nanoemulsions

Abstract

The purpose of this study was to prepare Brucea javanica oil cationic nanoemulsions (BJO-CN) with BJO as drug as well as oil phase and chitosan as cationic inducer, to explore the practical suitability of using cationic nanoemulsions for oral delivery of mixed oil, and to test its bioavailability and antitumor effect. BJO-CN was prepared by chitosan solution stirring method and then characterized physicochemically. The obtained BJO-CN had a spherical morphology with a positive zeta potential of 18.9 mV and an average particle size of 42.36 nm, showing high colloidal stability. The drug loading of BJO-CN was 91.83 mg·mL(-1), determined by high-performance liquid chromatography with precolumn derivatization. Pharmacokinetic studies revealed that, compared with BJO emulsion (BJO-E) (the dosage of BJO-CN and BJO-E was equal to 505 mg·kg(-1), calculated by oleic acid), BJO-CN exhibited a significant increase in the area under the plasma drug concentration-time curve over the period of 24 hours and relative bioavailability was 1.6-fold. Furthermore, the antitumor effect of BJO-CN in the orthotopic mouse model of lung cancer was evaluated by recording the median survival time and the weight of lung tissue with tumor, hematoxylin and eosin staining, and immunohistochemical technique. Results of anticancer experiments illustrated that, even though the administrated dosage in the BJO-CN group was half of that in the BJO-E group, BJO-CN exhibited similar antitumor effect to BJO-E. Moreover, BJO-CN had good synergistic effect in combination therapy with vinorelbine. These results suggested that cationic nanoemulsions are an effective and promising delivery system to enhance the oral bioavailability and anticancer effect of BJO.

Keywords: chitosan; combined chemotherapy; oleic acid; orthotopic mouse model of lung cancer; pharmacokinetics.

Figure 1

The pseudoternary phase diagrams of BJO-N. Notes: (A) K_m =3:1, RH40:Plu =2:1 (wt/wt); (B) K_m =3:1, RH40:Plu =4:1 (wt/wt); (C) K_m =3:2, RH40:Plu =3:2 (wt/wt); (D) K_m =2:1, RH40:Plu =4:1 (wt/wt); (E) K_m =1:1, RH40:Plu =2:1 (wt/wt). BJO was used as the oil phase. The black area represents the nanoemulsion existence range. Abbreviations: BJO-N, Brucea javanica oil nanoemulsions; K_m, the weight ratio of surfactant/cosurfactant; RH40, Cremophor RH40; Plu, Plurol Oleique CC497.

Figure 2

TEM analysis of BJO-CN. Note: Scale bar: 0.5 μm. Abbreviations: BJO-CN, Brucea javanica oil cationic nanoemulsions; TEM, transmission electron microscope.

Figure 3

Time-dependent change of particle size in BJO-CN. Notes: The blue line represents 4°C; the red line represents 25°C (n=3). Abbreviation: BJO-CN, Brucea javanica oil cationic nanoemulsions.

Figure 4

Time-dependent change of zeta potential in BJO-CN. Notes: The blue line represents 4°C; the red line represents 25°C (n=3). Abbreviation: BJO-CN, Brucea javanica oil cationic nanoemulsions.

Figure 5

Plasma concentration–time profiles of oleic acid in rats of the blank control group $\left(\mathrm{n}=8,\overline{x}\pm \mathrm{s}\right)$. Abbreviation: s, standard deviation.

Figure 6

Plasma concentration–time profiles of oleic acid in rats after oral single administration with BJO-CN, BJO-E $\left(\mathrm{n}=6,\overline{x}\pm \mathrm{s}\right)$. Abbreviations: BJO-CN, Brucea javanica oil cationic nanoemulsions; BJO-E, Brucea javanica oil emulsion; s, standard deviation.

Figure 7

CT image of lung tissues in cancer-bearing nude mice. Note: The red circles indicate the tumor. Abbreviation: CT, computed tomography.

Figure 8

Survival curves of nude mice after oral administration in all the groups: positive, combination, test, reference, and model group.

Figure 9

Pictures of lung tissues with tumor after oral administration in all the groups: positive, combination, test, reference, and model group. Note: The white block that appeared in the lungs was tumor tissue, and its visual volume was helpful to judge the growth of tumor.

Figure 10

Pathomorphological changes of A549 tumor section in all the groups (×100). Notes: (A) Positive group, (B) combination group, (C) test group, (D) reference group, and (E) model group.

Figure 11

Pathomorphological changes of A549 tumor section in all the groups (×400). Notes: (A) Positive group, (B) combination group, (C) test group, (D) reference group, and (E) model group.

Figure 12

Immunohistochemical staining for VEGF of orthotopic tumors in all the groups. Notes: (A) Positive group, (B) combination group, (C) test group, (D) reference group, and (E) model group. Magnification ×400. Abbreviation: VEGF, vascular endothelial growth factor.

Figure 13

Immunohistochemical staining for IL-8 of orthotopic tumors in all the groups. Notes: (A) Positive group, (B) combination group, (C) test group, (D) reference group, and (E) model group. Magnification ×400. Abbreviation: IL, interleukin.

Figure 14

Immunohistochemical staining for Bcl-2 of orthotopic tumors in all the groups. Notes: (A) Positive group, (B) combination group, (C) test group, (D) reference group, and (E) model group. Magnification ×400. Abbreviation: Bcl-2, B-cell lymphoma-2.

Figure 15

Immunohistochemical staining for P53 of orthotopic tumors in all the groups. Notes: (A) Positive group, (B) combination group, (C) test group, (D) reference group, and (E) model group. Magnification ×400.