Anti-Angiogenic and Anti-Cancer Evaluation of Betulin Nanoemulsion in Chicken Chorioallantoic Membrane and Skin Carcinoma in Balb/c Mice

Abstract

Betulin (Bet), the main component of birch tree bark, has been recently reported to exert anticancer activity in several cell lines; however the underlying mechanisms are only partially elucidated. The aims of the present work were to assess the in vivo effects of betulin administered as nanoemulsion (NE) in two experimental models: (i) the chicken embryo chorioallantoic membrane (CAM) assay for the study of anti-angiogenic effects and (ii) the two-stage model of skin carcinoma induced in mice for the study of anti-tumor and anti-inflammatory effects, respectively. On the CAM of the chicken betulin in nanoemulsion (BetNE) shows a good penetrability at extra-embryonic tissue level, affecting both the chorioallantoic membrane as well as the yolk sac by reducing the capillary density. In the animal model, the potential impact of local application of betulin on the respiratory function of isolated liver mitochondria was further assessed. Topical application of betulin nanoemulsion for 12 weeks together with DMBA (7,12-dimethylbenz[a]anthracene) and TPA (12-O-tetradecanoylphorbol 13-acetate), as tumor initiator and promoter, enhanced the active respiration of isolated liver mitochondria. Betulin also inhibit skin tumor apparition and promotion, proved by histological results and VEGF (vascular endothelial growth factor) expression correlated to non-invasive measurements. Betulin is active in nanoemulsion formulation as a potential inhibitory on the angiogenic process in CAM assay. BetNE can develop a potent anti-inflammatory and anti-carcinogenic activity with a low toxicity at skin level. It can also influence the penetration of carcinogens and reduce damage in main organs (e.g., liver).

Phytocompounds Targeting Cancer Angiogenesis Using the Chorioallantoic Membrane Assay

“…Immunohistochemical staining for smooth muscle actin (SMA) on the specimens treated with betulin in nanoemulsion, next Natural Products and Cancer Drug Discoveryto blank and control samples, are shown in Figure 3. The low expression of the marker in the betulin-treated specimen indicates a minimal implication of pericytes in the angiogenesis process [32]. On the contrary, we found that betulinic acid determined rapid maturation of the vessels and high levels of SMA [31].…”

“…Using the angiogenesis method in the rapid stage of CAM development, we found that pentacyclic triterpenes, betulinic (BA) acid, and betulin (Bet) in various formulations with cyclodextrin and in nanoemulsion are potential antiangiogenic compounds, acting differently, both through direct and indirect mechanisms [31,32]. Immunohistochemical staining for smooth muscle actin (SMA) on the specimens treated with betulin in nanoemulsion, next Natural Products and Cancer Drug Discoveryto blank and control samples, are shown in Figure 3.…”

Nanoemulsions in Translational Research—Opportunities and Challenges in Targeted Cancer Therapy

“…Betulinic acid is a pentacyclic triterpene which has the ability to target tumor cells and also vascular endothelial cells, thus functioning as an angiogenesis inhibitor. Dehelean et al recently assessed the in vivo anti-angiogenic and anti-inflammatory effects of betulinic nanoemulsion (8). Based on this work, it was identified that betulinic acid could be formulated in a nanoemulsion delivery system to attain potent anti-inflammatory activity with reduced exposure of the betulinic acid to vital organs.…”

“…In recent years, nanoemulsions, a heterogeneous liquid dispersion of nanoscale droplets of one liquid within another, have started evolving as a feasible carrier for the delivery of hydrophobic drugs. Their high solubilization capacity of hydrophobic drugs, ease of production, and long-term stability make nanoemulsions as promising drug delivery systems (2)(3)(4)(5)(6)(7)(8)(9). Nanoemulsion dosage forms have found wide applications in oral drug delivery to enhance the solubility and bioavailability of the hydrophobic drugs (4,10,11).…”

“…Nanoemulsion dosage forms have found wide applications in oral drug delivery to enhance the solubility and bioavailability of the hydrophobic drugs (4,10,11). Recently, there has been a surge in the exploration of nanoemulsions for parenteral drug delivery for the treatment of complex diseases such as cancer therapy (2,3,(5)(6)(7)(8)(9)12). The capacity of nanoemulsion to solubilize large amounts of hydrophobic drugs and their ability to protect the drugs from enzymatic degradation and hydrolysis make them ideal platform for the purpose of parenteral drug delivery.…”