Nano-delivery System Promotes Tumor Blood Coagulation And Microenvironment-Responsive Drug Delivery In Cancer Cells

It is a worldwide problem for the therapy of malignant tumors,which can be resected in the early stage,but mainly depend on chemotherapy in the late stage.Starvation therapy developed in recent years has achieved curative effect by reducing the sugar supply of cancer cells so as to starve tumors,but it is still challenging.Cancer cells are unable to be completely killed by only blocking the glucose supply because they resist to glucose deprivation-induced apoptosis in the presence of tumor lactic acid.Meanwhile,the blockage of blood vessels will stimulate the regeneration of tumor vasculature due to hypoxia.If the cancer cells cannot be quickly killed,the tumor blood vessels will be rebuilt.In this thesis,based on the strategy of starvation therapy and chemotherapy,we constructed a nano-drug carrier with induced tumor blood coagulation and p H/Hyals responsive drug delivery in cancer cells,for breast tumor therapy.The nano-carrier shows core-shell structure,good blood circulation stability and active targeting to breast cancer cells,as well as induced blood coagulation of breast tumor and p H/Hyals responsive drug release in cancer cells.Thus,it is a promising nano-delivery system against breast tumor.The detailed studies are as follows:?1?Synthesis and characterization of drug-loaded calcium carbonate nanoparticlesCalcium carbonate nanoparticles?Ca CO₃ NPs?were prepared by controlling the feed ratio of calcium chloride and sodium carbonate in an emulsifying system.XRD analysis confirmed the aragonite crystal form of Ca CO₃ NPs.The average particle size and PDI of Ca CO₃ NPs were measured to be 70.6 nm and 0.17 respectively by DLS.TEM observation showed that Ca CO₃ NPs were spherical and uniformly distributed.The average pore diameter of Ca CO₃ NPs was determined to be 7.81nm by BET.At an optimal mass feeding ratio of DOX:Ca CO₃ NPs=1:1,a DOX loading capacity of 49%was obtained with an encapsulation efficiency of 98%.In vitro DOX and Ca²⁺release tests showed that the release of both DOX and Ca²⁺was very slow in the environment mimicking blood circulation?p H7.4?,while in the environment mimicking endosomes/lysosomes?p H5.0?in cancer cell,about 90%of DOX was released within 48 hours and about 70%of Ca²⁺was released within 1 hour,indicating that the DOX and Ca²⁺releases of Ca CO₃ NPs were p H sensitive.Hemolysis test showed that in the concentration range of 0.01?2.0 mg/m L,the hemolysis rate of Ca CO₃ NPs was less than 2%,indicating good blood compatibility.The in vitro coagulation test showed that Ca CO₃ NPs did not cause blood coagulation at p H7.4,but induced rapid blood clotting at p H5.0,with a clotting time of 16 min.The in vitro uptake test showed that 74?80%of DOX@Ca CO₃ NPs were swallowed by MCF-7 and MDA-MB-231 breast cancer cells within 1 hour,and Ca²⁺and DOX,were fast delivered by acidolysis in endosomes/lysosomes,followed by the migration of Ca²⁺outside the cells and the diffusion of DOX into the nucleus.DOX@Ca CO₃NPs were significantly toxic to MCF-7 and MDA-MB-231 with an IC₅₀ value of 2.17?g/m L and 9.7?g/m L respectively,but they were non-toxic to mouse fibroblasts NIH3T3.The local injection of Ca CO₃ NPs in the breast tumor of mice showed that Ca CO₃ NPs could induce tumor vascular coagulation,moreover the higher the concentration of Ca CO₃ NPs,the more serious the blood coagulation.?2?Modification of hyaluronic acid by methacrylic anhydride and polyethylene glycolHA-PEG was synthesized by the reaction of the carboxyl group in HA with and amino group in methoxyl-and amino-activated polyethylene glycol?CH₃O-PEG-NH₂?.Then,m HA-PEG was obtained by the reaction between the 6-OH in HA and methacrylic anhydride?MA?.¹H-NMR analysis showed that the degree of substitution of PEG was 17.2%and that of MA was 22.9%.?3?Preparation and property of drug nano-carriers with core-shell structureDOX@Ca CO₃/m HA-PEG NPs with core-shell structure were prepared by the combination of DOX@Ca CO₃ NPs as the core and m HA-PEG as the shell,followed by ultraviolet radiation crosslinking.The average particle size and zeta potential of DOX@Ca CO₃/m HA-PEG NPs were measured to be 304.7 nm and-8.9 m V respectively by DLS.TEM observation showed that DOX@Ca CO₃/m HA-PEG NPs were spherical and uniformly distributed.TG test showed the content of core and shell occupied 70.6%and 29.4%respectively.DOX@Ca CO₃/m HA-PEG NPs presented a little change in particle size under the conditions of 0.05?0.30 M Na Cl and p H7.4,indicating their good stability in the environment mimicking blood circulation,However,the particle size significantly increased under the conditions of p H5.0 and Hyals,indicating that DOX@Ca CO₃/m HA-PEG NPs were unstable and p H/Hyals sensitive in the environment mimicking endosomes/lysosomes of cancer cells.In vitro DOX and Ca²⁺release tests showed that the delivery of both DOX and Ca²⁺was very slow in the environment mimicking blood circulation?p H7.4?,while in the environment mimicking endosomes/lysosomes?p H5.0?in cancer cell,about 80%of DOX was released within 48 hours and about 60%of Ca²⁺was released within 0.5hour,suggesting the p H/Hyals sensitive delivery of both DOX and Ca²⁺.The in vitro blood clotting test showed that DOX@Ca CO₃/m HA-PEG NPs did not cause blood coagulation at p H7.4,but the blood coagulation became faster as the acidity increased.A shortest clotting time of 12 min was achieved at the situation of p H5.0+Hyals.The in vitro uptake test showed that DOX@Ca CO₃/m HA-PEG NPs actively targeted to MCF-7 and MDA-MB-231,and were swallowed by these breast cancer cells via the specific interaction between CD44 and HA.In endosomes/lysosomes of cancer cells,DOX@Ca CO₃/m HA-PEG NPs were rapidly decomposed by p H/Hyals,leading to the delivery of DOX and Ca²⁺,such that DOX@Ca CO₃/m HA-PEG NPs efficiently killed both MCF-7 and MDA-MB-231 with an IC₅₀ value of 2.5?g/m L and 9.9?g/m L respectively,meanwhile,the delivered Ca²⁺migrated outward from the interior of cancer cells.