Preparation Of Thermo And PH Dual-responsive Nanoparticles And Mechanism Of Its Anti-stomach Tumor

Although traditional therapeutic methods for tumors have received considerable progress, the complete cure and eradication of cancer remain one of the greatest medical and health challenges at present. Nano technology based anti-tumor treatment has brought new hope for patients. Compared with traditional chemotherapy, drugs, wrapped in nanocarriers refered as nanomedicine, have the advantage of stability in vivo, low toxicity to normal tissues, controlled drug release and enhanced bioavailability. However, the development of nanomedicine systems based on traditional nanocarriers is not perfect yet. Greatly constrained by in vitro and in vivo conditions, the nanomedicine treatments were confronted with some bottleneck problems. Firstly, the stability of nanocarriers was still far from being applied in vivo and the circulation time in vivo was short. Secondly, the drugs accumulated in tumors were insufficient due to their poor targeting and instability. Thirdly, the endocytosis decreased because of weak targeting and binding of drugs to cancer cells. Fourthly, the release of drugs in specific intracellular sites was uncontrollable.In order to address these problems, we designed and synthesized a series of thermo- and p H-sensitive polymers. The micelles contained drugs were obtained from the dual responsive copolymer and drugs being dialyzed against water at p H 7.4. The drug delivery micelles consisted of thermo-sensitive N-isopropylacrylamide(PNIPAM) hydrophilic shell and p H-sensitive 2-(diisopropylamino) ethyl methacrylate(PDPA) core, with chemotherapy drugs doxorubicin(DOX) and/or amphotericin B(Am B) packed in the hydrophobic core. Drugs were accumulated in tumors by applying the differences between microenvironments of tumors and normal tissues. Intracellular nano-micelles immediately released drugs to cytoplasm due to the different p H among different organelles such as endosomes, autophagosomes and lysosomes, thus effectively inhibiting tumor growth. The following innovative results were mainly achieved:1. Using 4, 4’-Azobis-(4-cyanopentanoic acid)(V501) as initiator, 4-cyanopentanoic acid dithiobenzene(CPADB) as chain transfer agent, we synthesized a thermo-sensitive chain transfer agent PNIPAM using reversible addition-fragmentation chain transfer(RAFT) polymerization. IV Then four kinds of p H- and thermo-sensitive block copolymers with different chain lengths and different p H-sensitive blocks were synthesized by modifying the compositions of p H-sensitive monomer DPA and regulatory monomer 2-diethylamino ethyl methacrylate(DEAMEA), they were expressed respectively as PNIPAM114-PDPA70, PNIPAM114-PDPA140, PNIPAM114-PDPA140- PDEAEMA70, PNIPAM114-PDPA140-PDEAEMA140. Under acidic conditions whose p H=5, the volume phase transition temperatures(VPTT) of the above four block copolymers were 35.85℃, 35.87℃, 38.97℃ and 38.52℃ respectively. Under p H=8, the VPTT were 31.64℃, 30.72℃, 32.72℃ and 32.62℃. The p Ka values were 6.14, 5.93, 6.1, 6.26 respectively under 20℃ while they were 5.36, 5.18, 5.53, 5.77 under 39℃. The nano-micelles self-assembled by the copolymers in solutions could be regulated by altering the p H values of solutions, which resulted in the change of the block copolymer’s electrical charge. It revealed that the synthesized block copolymers got a favorable property of thermo- and p H-sensitivity. The block copolymers micelles packed with chemotherapeutic drugs were applied for cell proliferation inhibition. When the temperature increased, it facilitated the endocytosis of the micelles, thus enhancing killing of tumor cells. Meanwhile, we evaluated the effect of these four block copolymer loaded with si RNA under different temperatures. The results showed that as a gene carrier, the copolymer contained PDEAEMA block could lift transfection efficiency as the temperature elevated.2. The polymeric PNIPAM114-PDPA140 was selected for the subsequent in vivo experiments, whose VPTT was regulated to 39.1℃ by addition of m PEG. Its p Ka value was 5.9 under physiological conditions such as 0.15 mol/L Na Cl and 37℃. The size of the micelles was increased under both of temperature>39.1 ℃ and p H >5.9. However, the micelles were dissociation and distributed in the form of single copolymer when p H was lower than 5.9. The drug releasing profiles under different p H values and different temperatures were studied. The results turned out that with the p H decreased, the drug released more quickly. About 80% drugs released within 24 h when the p H was 5, while it reduced to less than 35% when the p H was 8. The drug release rate improved about two folds under 40℃compared with that under 37℃.3. The optimized nano-micelles wrapped with DOX were applied for tumor therapy. The killing effect raised double under 40℃. Relative to carriers wrapped with DOX under same temperature, the killing effect was 3-4 times higher with Am B and DOX simultaneously wrapped. Together with 3-MA, the formation of primary autophagosomes was inhibited, therefore suppressing nanocarriers within early endosomes, which in turn inhibited cell-killing effect of anti-tumor drugs. After Chloroquine(CQ) was introduced, the fusion of autophagosomes and lysosomes was stopped, thus the drug-loading nanoparticles were restrained in autophagosomes. Cell-penetrative Am B was released under low p H in autophagosomes, which was conducive for DOX to release to cytoplasm, avoiding drugs entering lysosomes and then being eliminated by lysosomes from the cell, thus enhancing killing effect on tumor cells.4. The tumor inhibiting effects of nanocarriers wrapped DOX, nanocarriers wrapped DOX and Am B, free DOX, and PBS were evaluated via tumor inhibition experiment in vivo. The results indicated that nanocarriers wrapped drugs were able to effectively accumulate inside tumors, therefore improving treating effect. Compared with PBS and free DOX, the inhibition effect of nanocarriers wrapped DOX were twice higher while the inhibition effect of nanocarriers simultaneously wrapped DOX and Am B were tripled. Therefore, the dual temperature and p H sensitive nanomedicine show high potent in tumor therapy and it has a guiding significance for clinical chemotherapy and antineoplastic carrier design.