Tumor Vessel Normalization By Metronomic Chemotherapy Of Neovasculature-Targeted Nanoparticulate Paclitaxel (F56-PTX-NP) And The Underlying Mechanism

Tumor vascular normalization is considered as a novel mechanism of antiangiogenic oncotherapy.During the“normalization window”typically tortuous and abnormal blood vessels transform to a more mature vessel structure,which results in homogeneous blood flow,reduced interstitial fluid pressure（IFP）and hypoxia.These alterations help to facilitate synergistic antitumor activity after the combination of antiangiogenic oncotherapy with chemotherapy or radiotherapy.Tumor vascular normalization may involve in the rebalance of many antiangiogenic factors and proangiogenic factors,and the detailed mechanisms remain to be further clarified.Currently,metronomic chemotherapy becomes a new modality of drug administration which involves the frequent administration of conventional chemotherapeutics at low dosage to target the tumor vascular endothelial cells.Metronomic chemotherapy can induce the secretion of TSP-1 which is important in tumor vascular normalization.Our previous study proved metronomic chemotherapy using nano-DDS could produce tumor specific biodistrbution,decreased off-target effects,and reduced accumulated dose.Thus it is very interesting to further investigate the tumor vasculature normalization using metronomic chemotherapy.In this study,F56-PTX-NP,paclitaxel loaded nanoparticles decorated with F56 peptide,was adoped as the model for metronomic chemotherapy.This study includes the following three parts:1.Preparation and characterization of F56-PTX-NP.F56-PTX-NP was engineered by emulsion and solvent evaporation method.The morphology and size of the nanoparticles were determined by TEM,AFM and DLS.The encapsulation efficiency（%）and drug loading（%）of paclitaxel were determined by HPLC.AFM and TEM images showed that F56-PTX-NP was spherical in shape with no aggregation.The nanoparticles had hydrodynamic diameters of 164 nm,encapsulation efficiency of33.7%,and drug loading of 2.9%.2.Internalization of nanoparticles and evaluation of its antiangiogenic activity.Nanoparticle internalization was assayed using High Content Analysis Reader and flow cytometry.Dynamic cell proliferation during metronomic treatment was monitored in real time using a RTCA instrument.HUVECs migration and tube formation were assayed to evaluate the antiangiogenic activity of F56-PTX-NP.F56conjugation dramatically facilitated nanoparticle uptake in HUVECs.Metronomic F56-PTX-NP led to significantly higher cytotoxicity and higher activity of inhibiting migration or tube formationto in HUVECs compared to other controls.3.Tumor vessel normalization by metronomic F56-PTX-NP and the underlying mechanism.Metronomic treatment was initiated by multiple dosing with a two-day break through the caudal vein.The tumors were removed and processed for sections and immunofluorescence staining for tumor vascular ECs,pericytes,and basement membrane.Then the tumor oxygenation,vascular permeability and perfusion were evaluated at day 16.The enhanced antitumor efficacy of conventional cytotoxic chemotherapy during the“normalization window”was assessed by tumor growth rate.A dramatical increase in the fraction of pericytes and basement membrane covered vessels were observed in the tumors treated by metronomic F56-PTX-NP from day 13to day 22.Metronomic F56-PTX-NP resulted in a remarkable increase in pO₂,decrease in hypoxia area,and higher tumor perfusion.Metronomic F56-PTX-NP plus DOX at day 16 significantly delayed the growth of MDA-MB-231 tumors compared with other controls.The aboved results suggested that metronomic chemotherapy using tumor-vessel targeted nanomedicine led to vascular normalization,and enhanced anticancer effects of conventional chemotherapeutics（DOX）administered during the induced normalization window.Our finding might provide new insight into the development of metronomic chemotherapy,tumor vessel normalization,and tumor vessel targeted nanoparticulate drug delivery system.