Long-circulating PCL-PEG Micelles With Enhanced Permeability Across The Tumor Extracelluar Matrix

Objective:This study aimed to design novel ? collagenase(Col?)and clusterin(CLU)modified PCL-PEG micelles loaded with doxorubicin(DOX),which can penetrate the tumor extracellular matrix(ECM)and inhibite macrophage phagocytosis,and to characterize the physicochemical properties of nanocarriers,and to investigate the in vitro drug release,safety,cytotoxicity,Penetration effect through ECM models,anti-phagocytosis ability,and anti-tumor effect of nanocarriers.Methods:PCL-PEG-Col? was synthesized by linking PCL-PEG and Col?through a carbodiimide method.DOX-loaded micelles(DOX-PCL-PEG-?)were self?assembly prepared,followed by noncovalantly adsorbing CLU on the surface of DOX-PCL-PEG-Col IV to obtain DOX-PCL?PEG-Col ?/CLU micelles.The physicochemical properties of functionalized micelles were investigated by means of laser light seattering particle size analyzer,transmission dectron microscopy(TEM)and fourier transform infrared resonance spectroscopy(FT?IR).The drug loading,entrapment efficiency and drug release of micelles were determined by a dialysis method.The safety of drug-free micelles was evaluated by a MTT method.The cellular uptake and anti-phagocytosis ability of micelles in MCF-7 tumor cells and RAW264.7 cells were investigated by laser confocal microscopy and flow cytometry.The penetration ability of DOX-PCL-PEG-Col?/CLU micelles was individually evaluated in the two-dimensional and three-dimensional ECM models.The tissue distribution of the micelles in the nude mice was observed by a small aninal imaging system,and the anti-tumor effect of DOX-PCL-PEG-Col?/CLU micelles was evaluated in MCF-7 cell-bearing nude mice.The effects of DOX-PCL-PEG-Col?/CLU micelles on tissues and organs were investigated by a HE staining method.Results:The results of physicochemical characterization showed that DOX-PCL-PEG-Col?/CLU micelles were successfully prepared.The drug loading and encapsulation efficiency of DOX-PCL-PEG?Col?/CLU micelles were 8.84%and 88.36%,respectively.The:in vitro release of DOX-PCL-PEG-Col?/CLU micelles in pH5.5 medium(62.98%)was higher than that in pH7.4 medium(11.88%).The results of MTT study showed that the cell survival rate of PCL-PEG-Col?/CLU was higher than 80%in 293T cells,RAW264.7 cells and MCF-7 cells,showing low cytotoxicity of blalk nanocarriers.The IC50 value of DOX-PCL-PEG-Col?/CLU micelles was 13.31 ?g/mL in MCF-7 cells.DOX-PCL.PEG?Col ?/CLU micelles could effectively prevent the phagocytosis of macrophage and show the good cellular uptake in MCF-7 cells analized by confocal laser microscopy and flow cytometry.Two.dimensional and Uiree-dimensional ECM models were estabilished for penetration experiments.The micelles modified with Col ? could effectively increase the penetration of nanocaririers across the ECM.DOX-PCL-PEG-Col?/CLU micelles could significantly reduce the distribution of drug in the liver and spleen,and enhance the drug accumulation in tumor tissue when compared to DOX-PCL-PEG-COOH micelles and DOX-PCL-PEG-Col ? micelles.A remarkable anti-tumor effect was observed for DOX-PCL.PEG-Col ?/CLU micelles with the inhibition rate of 85.62%.The results of HE staining indicated that DOX-PCL-PEG-Col?/CLU micelles did not cause pathological damages on main tissues including heart,liver,spleen,lung and kidney,and the caseous necrosis and transformation to fat cells occured in tumor tissues.Conclusion:Novel DOX-PCL-PEG-Col ?/CLU micelles had been successfully constructed.DOX-PCL-PEG-Col?/CLU micelles showed high safety,excellent cellular uptake in MCF-7 cells,penetration ability through the ECM,anti-phagocytosis ability and anti-tumor effects both in vivo and in vitro.This study provided theoretical basis for the study of long circulating nanocarriers to overcome the tumor ECM barriers.