Construction Of An Enhanced Hypoxia Activated Prodrug Responsive Nanosystem And Antitumor Study

Breast cancer is a malignant tumor disease that seriously threatens human health.Its biological properties of rapid proliferation and invasiveness,resulting in intratumoral hypoxia and adaptive antioxidation are common manifestations of breast cancer.Despite advances in therapeutic approaches,breast cancer treatment remains an urgent problem.In recent decades,novel strategies for photodynamic therapy,hypoxia inducible drugs,gene therapy as well as synergistic therapy have been gradually developed to target the tumor microenvironment and improve cancer treatment efficacy.Photodynamic therapy(PDT)is a non-invasive cancer treatment method with few side effects.However,the supply of photosensitizers,light and oxygen in tumor cells is insufficient,which makes its therapeutic efficacy still many constraints under complex tumor microenvironment.Gene therapy is a promising means of tumor therapy using therapeutic nucleic acids to treat diseases,but inefficient intracellular delivery of nucleic acids hampers their practical biological applications.Hypoxia inducing drugs are tumor treatment regimens that can selectively kill hypoxic cells and exhibit low toxicity in normoxic sites.But this prodrug relies on hypoxic environment activation to enhance antitumor efficacy.In order to solve the above problems,achieve efficient delivery of photosensitizers and antisense nucleic acids,enhance hypoxia at the tumor site to strengthen the therapeutic effect of hypoxia induced drugs,herein a nanodrug loaded system TPZ/ZIF-90@Ce6-G3139@HA(TZCG@HA)was constructed.Firstly,the nanosystem disintegrates under acidic conditions of tumor cells,releasing loaded antisense nucleic acids and photosensitizers.In which antisense nucleic acid can perform gene therapy,downregulating the expression of anti-apoptotic protein BCL-2 and accelerating cell apoptosis.On the other hand,photosensitizer can undergo photodynamic therapy,producing reactive oxygen species that interfere with the apoptosis mechanism of cells.While the continuous consumption of oxygen in PDT aggravates the hypoxic environment of the tumor,further activating the chemotherapeutic drug TPZ released in the acidic microenvironment of the tumor.The encapsulation of hyaluronic acid enables the nano drug delivery system to accurately locate drug release,achieving a combination of photodynamic therapy,gene therapy,and hypoxia inducible chemotherapy.The main experimental results are as follows: Based on the ZIF-90 multifunctional therapeutic nano platform,tumor microenvironment responsive nano drugs were prepared to achieve photodynamically induced hypoxia activated prodrug TPZ under near-infrared irradiation and combined gene therapy for breast cancer.At first,the constructed nano drug delivery system was characterized by TEM,particle size,potential and XRD data,and the reasonable usage of nanocarriers was also investigated.The encapsulation efficiency and drug loading of chemotherapy drug TPZ were calculated to be 45.98% and 19.67%,respectively.Meanwhile according to the antisense nucleic acid standard curve,it was concluded that the maximum adsorption amount of antisense nucleic acid G3139 was in the range of 180～200n M.The cumulative release of TPZ and G3139 from the nanocarriers was 73.72% and64.83%,respectively,under simulated tumor acidic microenvironment conditions.Then the in vitro cytotoxicity of the nanodrug loaded systems was examined,such as MTT,live dead cell staining and apoptosis,and the results showed that after the nanoparticles entered the MCF-7/ADM cells,the cytotoxicity of the group in the synergistic treatment of chemotherapy and gene therapy was stronger than that of the single drug loaded nanodrug system,while in combination with PDT treatment,the cell viability was inhibited to about 30%,showing a good cell killing effect.Meanwhile,laser confocal microscopy imaging demonstrated that the nanodrug loaded system had mostly been taken up by cells after 12 h incubation with cells.The intracellular ROS generation results showed that the drug loaded systems were able to generate more ROS after receiving light compared to the non-light group.The results of protein immunoblotting experiments indicated that nano systems could downregulate anti-apoptotic protein(BCL-2)and upregulate hypoxia inducible factor-1(HIF-1α)and cytochrome P450 reductase expression.Finally,the constructed in vivo mouse tumor model verified that the antitumor activity of the nano drug delivery system in vivo was consistent with the results of cell experiments,and during the treatment process,the weight of mice did not significantly decrease,indicating that the nanoparticles had good biocompatibility and had no significant toxicity to mice.This was confirmed by the H&E staining of the main organs of mice.In summary,this study successfully constructed TZCG@HA nanoparticles with good biocompatibility and good anti-tumor effects in vitro and in vivo.This strategy of utilizing PDT/hypoxia induced chemotherapy/gene therapy to synergistically treat breast cancer provides a novel idea to achieve highly efficient cancer treatment.