Pro-nifuroxazide Self-Assembly Leads to Triggerable Nanomedicine for Anti-Cancer Therapy.

Transcription factor STAT3 has been shown to regulate genes that are involved in stem cell self-renewal and thus represents a novel therapeutic target of great biological significance. However, many small molecule agents with potential effects in cancer therapy lack aqueous solubility and high off-target toxicity, hence impeding efficient bioavailability and activity. This work, for the first time, reports a prodrug-based strategy for selective and safer delivery of STAT3 inhibitors designed towards metastatic and drug resistant breast cancer. We have synthesized a novel lipase-labile SN-2 phospholipids-pro-drug from a clinically investigated STAT3 inhibitor, nifuroxazide (Pro-nifuroxazide), which can be regioselectively cleaved by the membrane abundant enzymes in cancer cells. Pro-nifuroxazide self-assembled to sub 20 nm nanoparticles and the cytotoxic ability was screened in ER (+)-MCF-7 and ER(-)-MD-MB231 cells at 48-72h using MTT proliferation assay. Results indicated that pro-nifuroxazide NP are multifold more effective towards inhibiting cancer cells in a time dependent manner compared to parent nifuroxazide. A remarkable improvement in the local concentration of drug to as high as ~240 folds when assembled into nanoparticles is presumably the reason for this functional improvement. We introduced molecular dynamics (MD) simulations to generate pro-nifuroxazidePro-nifuroxazide nano-assembly, a model assembly from triggerable anti-cancer drug, to provide molecular insights correlating physico-chemical and anti-cancer properties. In silico properties of pro-nifuroxazidePro-nifuroxazide including size and chemistry of nanoparticles and membrane interactions with individual molecules could be validated by in vitro functional activities in cells of breast cancer origin. The in vivo anti-cancer efficiencies of pro-nifuroxazidePro-nifuroxazide nanoparticles in nude mice xenografts with MCF-7 revealed remarkable growth inhibition as high as 400% for pro nifuroxazidePro-nifuroxazide nanoparticle. Histopathological analysis corroborated these findings showing significantly high nuclear fragmentation and retracted cytoplasm. Immuno-staining on tumor section demonstrated significantly lower level of pSTAT-3 by pro-nifuroxazidePro-nifuroxazide nanoparticle treatment establishing the inhibition of STAT-3 phosphorylation. Our strategy for the first time proposes a translatable prodrug agent self-assembled into nanoparticles and demonstrate remarkable enhancement in IC50, induced apoptosis and reduced stem like cancer cell population through STAT-3 inhibition and reduced phosphorylation.