Innovative Betulin Nanosuspension exhibits enhanced anticancer activity in a Triple Negative Breast Cancer Cell line and Zebrafish angiogenesis model

We present a nanosuspension of betulin, a BCS class II anticancer drug, particularly effective against resistant breast cancer. As anticancer efficacy of betulin is hampered by poor aqueous solubility, a nanosuspension with surface area was considered to enhance efficacy. An innovative approach wherein the betulin nanosuspension is generated instantaneously in situ, by adding a betulin preconcentrate (BeTPC) comprising drug and excipients, to aqueous medium, is successfully demonstrated. The optimal BeTPC when added to isotonic dextrose solution instantaneously generated an in situ nanosuspension (BeTNS-15) with high precipitation efficiency (92.7 ± 1.21%), average particle size (383.74 ± 7.24 nm) and good stability as per ICH guidelines. TEM revealed elongated particles while DSC and XRD indicated partial amorphization. Significantly higher cytotoxicity of BeTNS-15 (IC50 38.44 µg/ml) compared to betulin (BetS) (IC50 69.54 µg/ml) in the resistant triple negative human breast cancer cell line MDA-MB-231, was attributed to high intracellular uptake confirmed by HPLC and Imaging Flow cytometry (IFC). IFC confirmed superior anti-cancer efficacy of BeTNS-15 mediated by mitochondrial membrane disruption and inhibition of the G0/G1 phase. BeTNS-15 also exhibited significantly greater anti-angiogenic efficacy (p < 0.05) in the zebrafish model confirming superior efficacy. Simplicity of the innovative in situ approach coupled with superior efficacy proposes BeTNS as an innovative and highly promising anticancer formulation.