A PH/ROS Cascade Responsive Prodrug Nanosystem Against Multidrug Resistance Tumors

Chemotherapy is the main method of clinical cancer treatment which has some shortcomings such as poor tumor selectivity and serious side effects.Drug delivery systems(DDS)has been widely explored for which can protect drugs from phagocytosis and degradation in the circulatory system,prolong the blood circulation time,and significantly improve the targeting efficiency of tumors,thus significantly improving the bioavailability of drugs and the efficacy of anti-tumor therapy.However,DDS also face the challenges of low drug penetration,poor cell uptake and incomplete drug release,which limit the application of DDS in anti-tumor therapy.The emerging stimulusresponsive DDS will remain "invisible" during circulation to avoid being cleared.Once these kinds of DDS are exposed to pH,ROS,enzyme variation,they will change their charges,structure or size for deeper tumor penetration,more cell endocytosis,and higher drug release.In addition,stimulus-responsive DDS can distinguish tumor tissue from healthy tissue by responding to tumor microenvironment so as to reduce the toxic and side effects.Firstly,we fabricated a pH/ROS cascade responsive prodrug nanomicells(denoted as PPHI@B/L)by utilizing two amphiphilic diblock copolymers(PEG-Hyd-PCL,PEIPCL)with the loading of ROS-generation agent β-Lapachone and ROS responsive prodrug(BDOX).The successful synthesis of PPHI@B/L was proved by TEM,DLS,HNMR,TOF-MS and other characterization technique.The results of TEM and DLS verified that the particle size of PPHI@B/L could be reduced through the pH-responsive cleavage of hydrazone bonds under the weak acidic conditions(pH=6.8).The results of HPLC verified that the BDOX could release the therapeutic drug DOX through ROS response activation in the presence of ROS which was much less than the ROS content of tumor cells.Moreover,it is proved that the empty vector(PPHI)has good blood compatibility and biosafety by co-incubating with blood cells and many kinds of cells.Secondly,the effect of PPHI@B/L on overcoming multidrug resistance and antitumor therapy was explored in vitro.Confocal and flow cytometry verified that the reduction of PPHI@B/L particle size caused by pH response could increase the deep infiltration of tumor and expand cell uptake.ROS probe DCFH-DA verified that Lap in PPHI@B/L could generate effective ROS under the induction of excessive NQO1 in tumor cells.And Lap also consumed ATP and inhibited ATP-dependent P-gp protein in the process of genarating ROS,which overcame the multidrug resistance of tumor cells.Subsequently,the mechanism of overcoming multidrug resistance of PPHI@B/L was explored by examining intracellular ATP level and observing by confocal microscope.In addition,it was proved by Annexin V-FITC/PI,Calcein AM/PI,and CCK-8 that PPHI@B/L could effectively overcome multidrug resistance,distinguish normal cells from tumor cells,reduce the toxicity and side effects and improve the therapeutic effect.Finally,the effect of PPHI@B/L on overcoming multidrug resistance and anti-tumor therapy was explored in vivo.PPHI@Di R,carrying fluorescent molecule Di R was synthesized and verified by in vivo fluorescence imaging in mice that PPHI@Di R could effectively prolong the circulation time in vivo and accumulate passively to the tumor site,and that PPHI@B/L had the best anti-swelling effect and good biosafety compared with other control groups by BALB/c 4T1 cell tumor model.Through the MCF-7ADR model of BALB/c nude mice,it was proved that PPHI@B/L could inhibit multidrug resistance and improve the therapeutic effect compared with DOX group.Therefore,PPHI@B/L is a promising nanoplatform for overcoming tumor multidrug resistance and pathological barriers to enhance drug delivery.