| As cancer seriously threatens people’s life and health,it has become a global problem that needs to be solved urgently.At present,surgical therapy is the common treatment for the early-stage tumors and drug chemotherapy is chosen for the late-stage tumors.However,chemotherapeutic drugs lack targeting to tumor cells,presenting severe toxicity and side effects to normal tissues and organs.In recent years,killing cancer cells by inducing intracellular calcium overload,and promoting cancer cell death by eliminating tumor lactic acid that inhibits the death of cancer cells due to glucose deficiency in starvation therapy,are two new ways for cancer therapy.In this thesis,based on the idea of the combination of calcium overload and lactic acid elimination,we construct a p H/Lipase dually-responsive Ca（OH）2 nanoparticle,which can induce tumor calcium overload and eliminate lactic acid for the cancer treatment.The detailed contents are as follows:（1）Preparation and performance of delivery system based on Ca（OH）2@CaCO3NPsIn this chapter,Ca（OH）2@CaCO3 NPs were prepared in a microemulsion system and characterized.The results showed that Ca（OH）2@CaCO3NPs were uniform multi-core/shell spherical nanoparticles with a particle size of 85 nm,and 53.91%CaCO3 and 43.65%Ca（OH）2 in mass persentage respectively.Compared with CaCO3NPs,Ca（OH）2@CaCO3 NPs exhibited a better effect on lactic acid elimination and a faster Ca2+release in response to acid.However,the Ca2+release rate of Ca（OH）2@CaCO3 NPs reached 51%under neutral conditions due to the dissolution of Ca（OH）2by water,which was disadvantageous to the blood circulation stability of the nanoparticles.The premature release of Ca2+also resulted in the poor hemocompatibility of Ca（OH）2@CaCO3 NPs.Therefore,this thesis attempted to stop the premature Ca2+release by coating technology.However,TG and XRD results showed that neither cross-linked hyaluronic acid（c HA）coating or phospholipid（PL）coating could avoid the dissolution of Ca（OH）2 in Ca（OH）2@CaCO3 NPs by water under neutral conditions.Therefore,it is necessary to explore a new preparation technique of calcium nanoparticles.（2）Preparation and performance of delivery system based on CUR-Ca（OH）2-OA/PL NPsIn this chapter,the curcumin（CUR）-loaded Ca（OH）2-OA NPs were prepared by an oil-water interface method,and then PL was coated on CUR-Ca（OH）2-OA NPs by a film hydration method to obtain CUR-Ca（OH）2-OA/PL NPs for the prevention of premature Ca2+release,meanwhile the loaded CUR may induce intracellular calcium overload.The results showed that CUR-Ca（OH）2-OA/PL NPs were uniform spherical nanoparticles with an average particle size of 65 nm,and the loaded CUR and Ca（OH）2were 1.00%and 16.71%,respectively.The particle size of CUR-Ca（OH）2-OA/PL NPs was basically unchanged in normal saline,PBS and culture medium with 10%fetal bovine serum（FBS）within 48 hours,indicating good blood circulation stability.The p H value of phosphate buffer containing lactic acid was not affected by Ca（OH）2-OA/PL NPs（0～1 mg/m L）.However,in the presence of lipase,the p H value of phosphate buffer gradually increased with the increase of concentration,indicating the lactic acid elimination of Ca（OH）2-OA/PL NPs was lipase responsive.Nanoparticles released only 10%of Ca2+within 48 hours at p H 7.4,indicating that the PL coating successfully protected Ca（OH）2 in the nanoparticles from premature loss by water dissolution.Under the conditions of p H5.0+Lipase,the release of Ca2+and CUR reached 71%and 80%within 48 hours,indicating that the nanoparticles were p H/Lipase dually responsive.The hemolysis rate of Ca（OH）2-OA/PL NPs was less than5%,indicating good blood compatibility.The intracellular Ca2+concentration test showed that Ca（OH）2-OA/PL NPs and CUR-Ca（OH）2-OA/PL NPs increased the intracellular Ca2+fluorescence intensity from6.1 to 48.8 and 89.7 respectively,indicating that the Ca2+and CUR rapidly released from nanoparticles induced intracellular calcium overload in MDA-MB-231 cells.The cell viability of L929,A549 and MDA-MB-231 cells treated by Ca（OH）2-OA/PL NPs（80μg/m L Ca（OH）2）were 77%,67%and 33%,respectively,indicating that Ca（OH）2-OA/PL NPs were non-toxic to mouse fibroblasts L929 and low toxic to A549 cells,but they were significantly toxic to MDA-MB-231 cells.The IC50 value of CUR-Ca（OH）2-OA/PL NPs to MDA-MB-231 cells was 2.6μg/m L,which was lower than those of CUR-Ca（OH）2-OA NPs(IC50=4.4μg/m L)and pure CUR(IC50=7.7μg/m L).These results suggested that CUR-Ca（OH）2-OA/PL NPs have a better killing ability to MDA-MB-231 cells through lipase response and calcium overload.The effect of in vitro lactic acid elimination on cell viability showed after lactic acid elimination by Ca（OH）2-OA/PL NPs,the survival rate of MDA-MB-231 cells decreased from 9%to 2.2%.These results suggested that Ca（OH）2-OA/PL NPs could remove the inhibition of lactic acid on the death of cancer cells due to glucose deficiency,such that induce cancer cells death.In this thesis,p H/Lipase dually responsive nanoparticles（CUR-Ca（OH）2-OA/PL NPs）were successfully prepared to induce intracellular calcium overload and eliminate lactic acid,which provided a novel and potential way for cancer therapy. |
