Preparation Of PH-senstive“Nanobombs” For Researching Of Antitumor Activity

Multidrug resistance(MDR) remains a significant obstacle for the effective treatment of cancer disease. To address this formidable challenge, diverse cancer therapies have been developed to overcome the MDR of cancer cells efficiently. Christian de Duve, who was awarded the Nobel Prize in1974, defined firstly lysosomes as ‘‘suicide bags’ ’ that can cause cell and tissue autolysis upon rupture. Lysosomal membrane permeabilization(LMP) and subsequent cell death has been proven usefuL in cancer treatment. As LMP occurs, the release of certain cathepsins from the lysosome into the cytoplasm is thought to trigger cell death by apoptosis and apoptosis-like pathways. For cancer treament, this way can effectively overcome MDR, these organelles have gained much attention as potential therapeutic targets.In this work, hollow mesoporous silica nanoparticles employed as carrier to load anti-cancer drug doxorubicin, then simply treated by sodium bicarbonate to obtain the novel nano delivery system — — Nanobombs(NBs). We found that sodium bicarbonate can improve the rate of loading drugs; reduce the leakage of drugs under normal physiological conditions and improve its release rate under acid condition; more importantly, significantly improve therapy effect of cancer cells and resistant cancer cells. We also confirmed that the improvement of therapy effect was related with bicarbonate. NBs reacted with H+ in lysosome after endocytosed by cancer cell,quickly generate a large amount of CO2 bubbles, leading to lysosomal membrane permeability enhanced, inducing cell apoptosis eventually. Just a simple chemical process could significantly improve the performance of nano drug delivery system, effectively overcoming the drug resistance of cancer cells, it is a significant guide for designing a new drug delivery system.Concrete research content is as follows:(1) The construction of NBs. Silica microspheres was synthesized by modified Stober mothed, the collected nanoparticles were dispersed into Na2CO3 aqueous solution(0.2 M) for 2h at 80℃, The C18 TMS was removed by calcination at 550°C for 6 h. The obtain HMSNsexhibited a highly uniform and monodispersed spherical morphology. For the preparation of DMSNs, simply mixing HMSNs with doxorubicin. To obtain NBs, DMSNs was dispersed into NaHCO3 aqueous solution. solution and then treating with sodium bicarbonate. We charactered DMSNs, NBs with TEM images, SEM images, ultrasonic imaging and vitro releasing. The experiment showed that sodium bicarbonate could obviously improve the rate of loading drug.Compared with DMSNs, NBs in pH 7.4(simulating normal physiological environment)conditions, drug leakage was lower; While in pH 5.0(simulating lysosomal environment)conditions, the drug release rate was higher(53%), and release was more quick. Ultrasonic imaging experiments found that NBs produces a large amount of CO2 bubbles in pH 5.0condition. When NBs into lysosomes of cells, generation of CO2 bubbles prompted rapid release of DOX.(2) The research of NBs as drug dilivery system for the treatment of human lung cancer cells(A549). Free DOX, DMSNs, or NBs at a series of concentration incubated with A549 cells for 24 or 48 h, Cell viability was evaluated by the CCK-8 assay. Whatever 24 h or 48 h, the results showed the NBs had the most toxicity. Free DOX, DMSNs, or NBs at the same concentration incubated with A549 cells for different time, NBs also had the most toxicity. After Free DOX, DMSNs, NBs incubated with A549 cells, the morphology of A549 cell was real-time monitored by shooting video. The result indicated that the morphology of A549 cell changed firstly after incubating with NBs, with shrinking, turning round and shedding, these phenomena are signs of apoptosis. Confocal laser scanning microscopy(CLSM) to visualize DOX intracellular localization in A549 cells incubated with Free DOX, DMSNs, or NBs. For NBs, the red fluorescence mostly accumulated in the lysosomes, indicating that little DOX got into the MCF-7 cell nuclei. Due to NBs could generate a lot of CO2 under acidic conditions, we guessed that NBs could induced apoptosis by damaging lysosomal membrane. Lysosome imaging found the lysosome membrane of cancers cells suffered damage incabuted with NBs, a series of conclusions powerfully confirmed that NBs could induce apoptosis by enhancing lysosome membrane permeability.(3) The research of NBs as drug delivery system for the treatment of Michigan Cancer Foundation-7(MCF-7) and Michigan Cancer Foundation-7/drug resistant(MCF-7/ADR). The research of drug delivery system for the treatment of MCF-7. Free DOX, DMSNs, or NBs at a series of concentration incubated with MCF-7 cells for 24 or 48 h, Cell viability was examined by the CCK-8 assay. The results also showed the NBs had the most toxicity. CLSM was used to visualize DOX intracellular localization in MCF-7 cells after incubating with Free DOX,DMSNs, or NBs. For NBs, the red fluorescence mostly accumulated in the lysosomes, indicatingthat little DOX got into the MCF-7 cell nuclei. Lysosome imaging found the lysosome membrane of cancers cells incabuted with NBs suffered damage. The result of Caspase-3 activity showed NBs could enhance LMP, Increased LMP may cause the release of certain cathepsins from the lysosome into the cytoplasm, and can trigger death by apoptosis and apoptosis-like pathways. NBs can kill cancer cells by targeting lysosomes without the aid of DOX, which avoid the drug efflux. and as such, NBs should be able to overcome the MDR of cancer cells. Finally,Free DOX, DMSNs, or NBs at a series of concentration incubated with MCF-7/ADR cells for 24 or 48 h, Cell viability was assessed by the CCK-8 assay, the data showed that NBs can effectively overcome the drug resistance.