Endothelial-specific nanodelivery of Rho kinase inhibitors: Targeting tumor angiogenesis and metastasis

OBJECTIVE: The limitations of current vascular endothelial growth factor (VEGF)-centered anti-angiogenic therapy include the inhibition of physiological angiogenesis, development of evasive resistance, and increased vascular leakage, which drives the need for a novel therapy for tumor angiogenesis and metastasis. RhoA pathway inhibition blocks VEGF- and sphingosine-1 phosphate (S1P)-induced angiogenesis and vascular leakage without hindering endogenous angiogenesis. Fasudil, a clinically relevant Rho-associated kinase (ROCK) inhibitor, blocks the RhoA downstream signaling, but adverse events (hypotension, skin reactions) exist. Here, we propose endothelial ROCK targeting with Fasudil-containing immune-related nanodelivery. HYPOTHESIS: Fasudil-encapsulated CD31-targeting liposomal formulation is a potential tumor antiangiogenic therapy by blocking RhoA signaling via ROCK inhibition in the endothelial cells. METHODS: Fasudil-liposomal nanoparticles were prepared by DPPC, cholesterol, 16:0 PEG2000PE, DSPE-PEG2000-mal, DSPE-PEG-FITC, and CHCl3: MeOH (4:1) combination, thin-film formation, downsizing after Fasudil incorporation, filtering, and conjugation with IgG and CD31-targeting antibodies. We used dynamic light scattering (DLS) for the liposomal characterization, dose-response experiments on primary Human Umbilical Vein Endothelial Cells (HUVEC) and Bend3 cells for in vitro toxicity evaluation, antibody (Ab)-to-liposome binding evaluation and pathway inhibition evaluation by western blot, and UPLC analysis (EE)% for the evaluation of encapsulation efficiency. The binding affinity of the CD31-conjugated liposomes and their time-dependent cell incorporation were evaluated by competitive binding assay via immunohistochemistry (IHC) and flow cytometry. Morphometric analysis was assessed by Livecyte imaging analysis. RESULTS: Size and PDI measurement by DLS confirmed the stability of our formulation, which presented limited toxicity (concentration: 0.625-5120 µM; IC50 at 43.05 µM [HUVECs over 24 hours of treatment]) with a significantly higher overall survival rate than Fasudil. Efficient CD31 and mouse IgG Ab conjugation to Fasudil liposomes and blockade of the RhoA downstream signaling in endothelial cells were confirmed. The specific binding of CD31-targeting liposomes on endothelial cells was verified in the presence and absence of excess CD31 Ab. The time-dependent study confirmed the kinetics of liposome incorporation in endothelial cells and the enhanced uptake of CD31 liposomes compared to IgG. Cell morphometric and physiological analysis revealed comparable alterations caused by Fasudil- and endothelial-specific Fasudil-containing liposomes. CONCLUSION: We have successfully developed Fasudil-encapsulated, CD31-targeting liposomes. Future studies will be directed to study the formulation's effect on endothelial physiology in vitro and in vivo, tumor growth, angiogenesis, and metastasis. © FASEB.