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. 2019 May 22;11(20):18074-18089.
doi: 10.1021/acsami.9b01343. Epub 2019 May 13.

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

Santosh K Misra  1   2 , Zhe Wu, Fatemeh Ostadhossein  1   2 , Mao Ye  1   2 , Kingsley Boateng, Klaus Schulten, Emad Tajkhorshid, Dipanjan Pan  1   2
Affiliations

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

Santosh K Misra et al. ACS Appl Mater Interfaces. .

Abstract

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 ∼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.

Keywords: cancer therapy; dissipative particle dynamics; nanoparticle; prodrug; self-assembly.

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Figures

Figure 1.
Figure 1.
Pro-nifuroxazide nanoparticle structure resulted from coarse-grained (CG) dissipative particle dynamics (DPD) simulations of the self-assembly process. (A) CG representation of Pro-nifuroxazide. (B) Steps for the simulations before the self-assembly of Pro-nifuroxazide molecules. (C) A representative structure of a Pro-nifuroxazide nanoparticle. (D) Cross-section of the Pro-nifuroxazide nanoparticle. Color code: hydrophobic carbon chain of PEGCE (yellow), hydrophilic polyethylene glycolic part of PEGCE (blue), carbon chain of PAzPC in outer shell (green), PAzPC head group (light blue), and nifuroxazide moiety (orange).
Figure 2.
Figure 2.
Physicochemical characterization of different chemistries and formulations of Nifuroxazide. (A-B) Anhydrous morphology of Pro-nifuroxazide nanoparticles (NPs) obtained using transmission electron microscopy in negative staining mode (TEM, uranyl acetate) from different section of electron grids. (C) Hydrodynamic diameter of Pro-nifuroxazide NP and (D) stability of the self-assembled Pro-nifuroxazide nanoparticle (pH 7.4). (E) UV-vis spectrum of free nifuroxazide molecules in water revealing characteristic absorbance of the drug molecule. (F) Zeta potential; (G) nuclear magnetic resonance (NMR) spectra of the Pro-nifuroxazide (a) 1H and (b) 13C traces (CDCl3).
Figure 3.
Figure 3.
(A) Schematic representation of a prodrug nanoparticles interacting with cell membrane and cross section of simulated Pro-nifuroxazide nanoparticle structure with segregation of various components of the Pro-nifuroxazide and PEGCE molecules. (B) Conformations of Pro-nifuroxazide as it approaches the membrane. Distances below the images indicate the position of the nifuroxazide group relative to the membrane interface (z=0). Strong interaction of the PAzPC lipid tail with membrane increases the prodrug-membrane binding range and affinity. (C) Potential of mean force curves for individual nifuroxazide (black) or Pro-nifuroxazide (red) molecules inserting into a POPC membrane. The reaction coordinate (the abcissa) is the distance between center of mass of the nifuroxazide moiety and membrane interface. The free energy curves were calculated from −15 to 35 Å, that is, a range between close positioning of the drug moiety to the membrane center at z = −20 Å, and its complete dissociation from the membrane (35 Å).
Figure 4.
Figure 4.
MTT assay performed on (A) MDA-MB-231 and (B) MCF-7 cells after 72 h treatment of nifuroxazide and Pro-nifuroxazide at concentrations ranging from 0.5 to 20 μM and (C) comparison of IC50 values; (D) selective low response of Pro-nifuroxazide nanoparticles demonstrated in non-cancerous MCF-10A breast cells; (E) summary of apoptotic cell population (20 μM). Biostatistical analysis was performed using ONE Way ANOVA with post Bonferroni test. Here *, ** and *** represent p values <0.05, 0.01 and 0.001, respectively.
Figure 5.
Figure 5.
Cell internalization mechanism of Pro-Nifuroxazide NPs. (A) Inhibitor pre-incubation study performed on MCF-7 cells after Pro-Nifuroxazide treatment at 20, 10 and 5 μM concentration of Nifuroxazide or Pro-Nifuroxazide NPs alone. (B-D) Cell TEM performed on I-Pro-nifuroxazide NPs) after 30 min of incubation showing position of membrane fusion as mode of cellular entry. Cell incubated with rhodamine alone (E, F) and Rh-Pro-nifuroxazide NPs (G, H). Here E and G represent DAPI stained cellular nucleus while F and H shows Rh distributed in intracellular space. Cells were incubated with rhodamine and Rh-Pro-nifuroxazide NPs for 4h. Here * represents p values <0.05 and 0.001, respectively.
Figure 6.
Figure 6.
(A) STAT-3 inhibition study in MDA-MB231 cells at 48h incubation time point. Nifuroxazide was used in form of small molecule, Pro-nifuroxazide and prodrug nanoparticle at a concentration of 20 μM. Cells were incubated for 48h before collecting the RNA and performing PCR studies. Pro-nifuroxazide nanoparticle showed maximum inhibition in STAT-3 expression followed by Pro-nifuroxazide while nifuroxazide showed the minimum inhibition. (B) STAT-3 protein inhibition study in MDA-MB231 cells at 48h incubation time point. Nifuroxazide, Pro-nifuroxazide and Pro-nifuroxazide nanoparticle at final concentration of 20 μM were used. Cells were incubated for 48h before collecting the total protein and performing Western Blot studies (C). Pro-nifuroxazide nanoparticle showed maximum inhibition in STAT-3 expression followed by proPro-nifuroxazide while nifuroxazide alone showed the minimum inhibition. Biostatistical analysis was performed using ONE Way ANOVA with post Bonferroni test. Here * represents p values <0.05 and 0.001, respectively.
Figure 7.
Figure 7.
In vivo evaluation of tumor regression by treating xenograft tumors generated from MCF-7 cells grown in nude mice animal models. (A) Tumors were grown to a minimum size of 0.5×0.5 cm2 before injecting with phosphate buffer or Pro-nifuroxazide nanoparticles in 40 μL volume on day 0, 4, 8 and 12. Tumors were monitored for a total of 28 days (B, D) before sacrificing and (C, E) collected representative tumor tissue from animals treated with (B, C) phosphate buffer and (D, E) Pro-nifuroxazide nanoparticles on day 28. Analysis of (F) % tumor growth and (G) % effective growth inhibition across experimental time line for buffer and Pro-nifuroxazide NP treated animals. H&E staining on tumor sections from animals treated with (H, I) phosphate buffer and (J, K) Pro-nifuroxazide nanoparticles. Arrows represent areas of cellular degenerations in tumor sections from Pro-nifuroxazide nanoparticle treated animals. Biostatistical analysis was performed using ONE Way ANOVA with post Bonferroni test. Here * and *** represent p values <0.05 and 0.001, respectively.
Figure 8.
Figure 8.
Representative immune-labeled cross sections of tumors treated with phosphate buffer saline (A-C) and prodrug nanoparticles (D-F). Sections were incubated with pSTAT-3 antibody (red) and background protein β-actin (green) around cell nuclei stained with DAPI (blue) to show significantly high level of pSTAT-3 in phosphate buffer saline treated tumors compared to treated with Pro-nifuroxazide nanoparticles.
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