| Clinical oncology treatments often fail due to systemic toxicity of therapeutic agents and the complex tumor microenvironment.Numerous studies have demonstrated that nano-carriers,especially nano-liposomes can reduce the systemic toxicity of therapeutic agents and improve therapeutic efficacy.However,the intricacies of disturbing factors in the tumor microenvironment,including complex physiological barriers(e.g.,abnormal vascular networks,increased tissue fluid pressure,and abundant extracellular matrix)and cellular factors(e.g.,high drug efflux capacity and elevated levels of anti-apoptotic),lead to high probability of metastasis and recurrence in cancer patients even after treatment.Although advanced nanocarriers based on inorganic,organic and hybrid nanomaterials have been extensively investigated to address drug resistance,few suitable strategies have so far been able to fully address the aforementioned challenges.To overcome these obstacles,synergistic therapy with gaseous signaling molecules such as oxygen(O2),nitric oxide(NO),carbon monoxide(CO),hydrogen sulfide(H2S)and hydrogen(H2)has gradually attracted the attention from researchers.Appropriate concentration of gas signaling molecules can diffuse freely into tumor cells and interstitial tissues without any active transport mechanism,and inhibit the survival of cancer cells without affecting the normal cells.However,it is still very challenging to deliver such gases with very short half-lives to the lesion.In this thesis,we have prepared liposomes which are sensitive to tumor microenvironment,encapsulated with gas signal molecule prodrug,using the surface modifiable and controlled loading structural features of liposome system,and systematically investigated and validated the efficiency of liposomes as intratumor microbubble drug delivery system using subcutaneous graft tumor and in situ glioma mouse as animal models,the results of multimodality enhanced imaging and tumor tissue therapy were evaluated.The specific research content included the following four parts:(1)Liposomes(AMLs)loaded with anisotrisulfide(ADT)drugs and magnetic nanoparticles were prepared and the the mechanism interaction with cells were studied.1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine(DPPC),1,2-distearoyl-sn-glycero-3phosphatidylcholine(DSPC),1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2000)phospholipid,superparamagnetic nanoparticles(SPIONs)and the ADT,H2S gas precursor,were used to prepare magnetically targeted drugcontaining nanoliposomes(AMLs)with a hydrodynamic size of 163.3±25.1 nm(PDI=0.196)by an extrusion permembrane method.The AMLs showed a sub-circular structure under transmission electron microscopy and could be stored for 1 month at 4℃.The saturation magnetization intensity of the AMLs was 72 emu/g,the encapsulation rate of the magnetic nanoparticles was 84.1±5.1%,and the encapsulation rate of the ADT drug was 49.6±3.4%.Further,AMLs were interacted with cells to evaluate their safety,targeting,image enhancement and therapeutic effects.Cytotoxicity assays of L-02 human normal hepatocytes and HepG-2 human hepatocellular carcinoma cells after co-incubation with AMLs showed that after 24 h of co-incubation,AMLs did not affect normal cell activity,while the inhibition rate of tumor cells exceeded 60%.After 2 h of 200 mT static magnetic field stimulation,AMLs accumulated in tumor cells in large quantities and enhanced the magnetic resonance T2 contrast imaging ability of tumor cells.With the release of ADT in tumor cells,H2S was generated by the action of specific cystathionine-β-synthase(CBS)ystathionine-γ-lyase(CSE)enzymes in tumor cells.At low concentration,H2S killed tumor cells through the destruction of redox environment and ion gradient in tumor cells;with the increase of concentration,H2S formed nano-to micron-scale bubbles in cells,which obviously enhanced the ultrasonography effect at cellular scale;finally,the generation of a large number of intracellular bubbles destroyed the tumor cytoskeleton,leading to the lysis and death of tumor cells.(2)The effect of AMLs on mice subcutaneous tumors modelThe safety,targeting and bimodal imaging effects of tail vein injection of AMLs were further evaluated in an animal model of HepG-2 subcutaneous tumors.The results of near-infrared fluorescence imaging in small animals demonstrated that the accumulation of AMLs in the tumor region after 4 h of injection under static magnetic field guidance was 2.34 times higher than that in the blank control group;the results of magnetic resonance and ultrasound imaging in small animals showed that the release of magnetic nanoparticles and ADT drugs significantly enhanced the magnetic resonance/ultrasound bimodality imaging in the tumor region after targeting of AMLs to the tumor;the major organs and tumor tissues H&E,Prussian blue and TUNEL staining results showed that AMLs did not damage normal tissues and organs,while the static magnetic field could induce AMLs to penetrate into the central tumor region and kill tumor tissues through the cavitation effect of ADT molecules after conversion into H2S bubbles,further enhancing the therapeutic effect.(3)The nanoliposomes(S1P/JS-K/Lipo)loaded with JS-K drug and targeting molecules were prepared and mechanism interaction with cells were studied.Using DPPC,DSPC and DSPE-PEG2000 as liposome backbones and sphingosine 1-phosphate(SIP)as target molecule,S1P/JS-K/Lipo with a hydrodynamic size of 322.6±41.93 nm(PDI=0.211)loaded with NO gas precursor drug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]azo1-yl was prepared by extrusion periplasmic method.This S1P/JS-K/Lipo has a sub-circular structure under TEM and can be stored at 4℃ for 1 month.In a solution simulating a high concentration of glutathione S-transferase(GST)enzyme environment in glioma cells at a concentration of 3.57 ng/mL,the release rate of JS-K drug from liposomes was 68%within 2 h and 81%within 8 h.Further,S1P/JS-K/Lipo was interacted with cells to investigate the mechanism of its effect on penetrating the blood-brain tumor barrier(BBTB)and killing glioma cells under the barrier.The cytotoxicity assay results showed that S1P/JS-K/Lipo had a good killing effect on U87MG glioma cells with low toxicity to vascular endothelial cells bEnd.3.Immunostaining confocal microscopic imaging showed that SIP/JS-K/Lipo activated caveolin-1(Caveolin-1)in the BBTB structure using SIP molecules,inhibited the expression of the cellular efflux pump P-glycoprotein(P-gp),and enhanced the ability of nanocarriers to transport across the BBTB.(4)Diagnostic and therapeutic applications of S1P/JS-K/Lipo in in situ glioma mice modelsThe targeting imaging and therapeutic effects of tail vein injection of S1P/JS-K/Lipo on glioma treatment were further evaluated in an animal model of in situ glioma.Small animal near-infrared fluorescence imaging experiments showed that S1P/JS-K/Lipo actively targeted the in situ glioma region within 4 h,and the cumulative effect within 24 h was twice as high as that of the untargeted experimental group;Laser scattered blood flow imaging and ultrasound imaging results showed that the therapeutic molecule JS-K delivered after S1P/JS-K/Lipo crossed the BBTB stimulated by glioma-specific GST enzymes,in situ NO gas was produced,which improved blood supply to the glioma area,normalized blood vessels,and enhanced ultrasound imaging of the glioma area;tissue section staining demonstrated that S1P/JS-K/Lipo inhibited the growth of glioma and prolonged the survival of rats with tumors. |
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