Pro-Nifuroxazide Self-Assembly Leads to Triggerable Nanomedicine for Anti-cancer Therapy

SK Misra and Z Wu and F Ostadhossein and M Ye and K Boateng and K Schulten and E Tajkhorshid and DPJ Pan, ACS APPLIED MATERIALS & INTERFACES, 11, 18074-18089 (2019).

DOI: 10.1021/acsami.9b01343

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 through STAT3 modulation 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 toward metastatic and drug- resistant breast cancer. We have synthesized a novel lipase-labile SN-2 phospholipid prodrug 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 (NPs), and the cytotoxic ability was screened in ER(+)-MCF-7 and ER(-)-MD-MB231 cells at 48-72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide proliferation assay. Results indicated that Pro-nifuroxazide NPs are multifold more effective toward inhibiting cancer cells in a time- dependent manner compared to parent nifuroxazide. A remarkable improvement in the local concentration of drugs to as high as similar to 240 fold when assembled into NPs is presumably the reason for this functional improvement. We also introduced molecular dynamics simulations to generate Pro-nifuroxazide nano-assembly, as a model assembly from triggerable anti-cancer drugs, to provide molecular insights correlating physicochemical and anti-cancer properties. In silico properties of Pro-nifuroxazide including size, chemistry of NPs 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-nifuroxazide NPs in nude mice xenografts with MCF-7 revealed remarkable growth inhibition of as high as 400%. Histopathological analysis corroborated these findings to show significantly high nuclear fragmentation and retracted cytoplasm. Immunostaining on tumor section demonstrated a significantly lower level of pSTAT-3 by Pro-nifuroxazide NP treatment, establishing the inhibition of STAT-3 phosphorylation. Our strategy for the first time proposes a translatable prodrug agent self-assembled into NPs and demonstrates remarkable enhancement in IC50, induced apoptosis, and reduced cancer cell population through STAT-3 inhibition via reduced phosphorylation.

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