Sequential Intercellular Delivery Nanosystem For Enhancing ROS-induced Anti-tumor Therapy

Cancer has become a serious problem of threatening human's health and life.Breaking the threshold of reactive oxygen species?ROS?level to induce irreversible oxidative damage has been extensively applied to cancer treatment.Photodynamic therapy?PDT?,a representative approach of ROS-based cancer therapies,which is largely due to many drawbacks such as oxygen dependence and limited light transmission in traditional PDT therapy hindered its clinical treatment efficiency.In order to improve PDT-mediated ROS therapy,the researchers developed a non-photodynamic system to induce cancer cell death by using exogenous ROS-agents that did not require oxygen and external stimuli.Although promising,these systems frequently neglect the fact that abnormal tumor microvessels and increased diffusion distances from cells to blood vessel induced insufficient oxygen supply while making drug or nanoparticles with little opportunity to reach the deep tumor cell.It is difficult to achieve effective therapeutic effect of ROS only by increasing the oxygen concentration in the tumor.Inspired by intercellular lipid transfer mediated by EVs,we propose a sequential intercellular delivery nanosystem for enhancing ROS-induced anti-tumor therapy,which is an artificial membrane fusion liposome?MFLs?as antitumor agent-carrier.In this subject,we selected the LAOOH,as one of phospholipid peroxide inherent in the cell membrane,which generated ROS catalyzed by metal ions(for example,Fe²⁺,Ce⁴⁺)rather than dependent on O₂ and other external stimuli,loading within the phospholipid bilayer of MFLs.And doxorubicin?DOX?,as the clinical model drug,wasencapsulatedintothehydrophiliccoreofMFLs,obtaining MFLs/LAOOH@DOX.Firstly,MFLs/LAOOH@DOX selectively fused with tumor cell membranes in the perivascular region via the enhanced permeation and retention effect?EPR?.Followed the intercellular lipids exchange mediated by EVs,LAOOH,as cell membrane natural ingredient,spread gradually from the outer layer tumor cells to neighboring cells closer to the center of the tumor and distribute throughout the entire tumor eventually.With the assistance of subsequent administration of nano Fe₃O₄,which was dissociated to Fe²⁺under tumor tissue or lysosome acidic pH environment,tumor membrane-locating LAOOH was catalyzed and produced abundant ROS through the Russell mechanism,resulting in tumor cells apoptosis and necrosis.Furthermore,DOX be efficiently delivered into nucleus of tumor cells accompanied by membrane fusion between MFLs and tumor cells,thus decreasing DOX degradation in lysosomes.In this subject,we successfully prepared nano-Fe₃O₄.TEM indicated that the Fe₃O₄ possessed good monodispersity and diameter is about 10 nm.Additionally,the results demonstrated that the degree of ferric ions released from Fe₃O₄ is a pH-dependent manner.Under pH 5.5 condition,the Fe₃O₄ showed 30%release of ferric ions for 48 h incubation.The analysis of LC-MS and Ultraviolet-visible spectroscopy showed that LAOOH was successfully prepared and its ROS performance show that LAOOH can be used as an effective ROS-agents.The MFLs/LAOOH@DOX was prepared by thin film dispersion method.Then,the MFLs/LAOOH@DOX was characterized by transmission electron microscopy?TEM?and dynamic light scattering and its release behavior was also observed.The TEM showed that it was a uniform spherical shape with a particle size of 70 nm and has a good stability at 4?.In the vitro drug release experiment,release of DOX in different formulations is dependent on the Fe²⁺.It indicates that the LAOOH in the phospholipid bilayer of MFLs can effectively produce ROS in the presence of Fe²⁺,and oxidize macromolecular phospholipids to destroy liposome bilayer,therefore triggered DOX burst release from the hydrophilic core of MFLs.We used the 4T1?mouse breast carcinoma?cell lines as a model cell.Fluorescence microscopy and flow cytometry were used to detect the ability of LAOOH with different concentrations to generate ROS in cell level.The results showed that the formations containing LAOOH only produced ROS in the presence of Fe₃O₄,and the ROS amount increased with the concentration of LAOOH.Simultaneously,we observed that the morphology of cells in MFLs/LAOOH adding Fe₃O₄ groups changed dramatically,which lost their original phenotype,whereas the cells treated with CLs/LAOOH did not show any distinct morphological change.These results implied that the LAOOH delivered by MFLs shown more cytotoxicity to tumor cells.Then we investigated the subcellular localization of MFLs in 4T1 cell lines to track the distribution of liposomes in tumor cell.The images revealed that MFLs delivered lipid mainly on the tumor cell membrane.Comparing MFLs/LAOOH@DOX with CLs/LAOOH@DOX,confocal microscopy images demonstrated that MFLs/LAOOH@DOX was internalized into tumor cells through the membrane fusing approach rather than endocytosis.The ability of different formations to induce tumor cells apoptosis was studied as well.The results showed that the MFLs/LAOOH mediated by membrane fusion method combined with Fe₃O₄could effectively induce apoptosis.Transwell experiments showed that MFLs can transfer LAOOH to neighboring cells via EVs-mediated intercellular lipid transfer,and ROS efficiently increased after co-incubation with Fe₃O₄.Subsequently,the 3D tumor sphere model experiment showed that MFLs was capable of increasing the tumor penetration depth.The zebrafish embryos results showed that MFLs can across the mucus-covered enveloping layer cells to the cells within the underlying epidermal basal layer and efficiently deliver therapeutic agent such as PI into cell nucleus.We used the 4T1 BALB/C tumor-bearing mice as models to investigate the therapy efficacy of MFLs-based formulations in-vivo.The tissue distribution of the MFLs in the tumor-bearing mice by NIR fluorescence imaging system showed that the MFLs indeed enhanced the tumor specific accumulation and retention through the EPR effect compared with sole DIR and accumulated at the highest level at 1 h.So the interval between an injection twice daily would be 1 h.In vivo tumor tissue penetration experiments,the MFLs-based nanosystem exhibited superior accumulation in both peripheral and interior regions.Accordingly,we also observed that ROS was produced substantially in the tumor of mice treated with MFLs/LAOOH+Fe₃O₄ compared with CLs/LAOOH+Fe₃O₄.These observations demonstrated that tumor cell membrane-selective delivery mediated by MFLs could enhance tumor deeper penetration of therapeutic agents and thus improve the antitumor treatment efficiency.Pharmacodynamic results showed that the anti-tumor effect was more significant after the second injection of Fe₃O₄,and the tumor growth inhibition rate was 82%.It indicates that the combination of Fe₃O₄ and MFLs/LAOOH@DOX can enhance the anti-tumor efficiency,and the anti-tumor effect of MFLs formations is greater than the CLs formations.The TUNEL was consistent with the in vivo pharmacodynamic results,further confirming the significant anti-tumor effect of MFLs/LAOOH@DOX+Fe₃O₄.It can be seen from the body weight changes and cardiac panoramic scan of different formations that MFLs/LAOOH@DOX significantly reduced the toxic effects of DOX.We selected the LAOOH as a ROS-agents,which generated ROS catalyzed by metal ions(for example,Fe²⁺,Ce⁴⁺)rather than dependent on O₂ and other external stimuli.Meanwhile,we constructed a MFLs-based sequential intercellular delivery system mediated by EVs,which directionally transfer ROS-agents between cells,like the random walk Brownian motion of molecules.This sequential intercellular delivery system made therapeutic agents effectively deliver to deep tumor tissue and produced ROS onto the tumor cell membrane to enhance ROS-induced antitumor efficacy through improving both ROS productivity and tumor distribution,which provided a new idea for the future clinical treatment of cancer.