Dual-targeted Nanodrug-guided Radiotherapy Enhances DNA Lesion Formation And Inhibits DNA Damage Response In Breast Cancer

Objective:Both production of DNA damage and subsequent prevention of its repair are crucial in concluding the therapeutic outcome of radiotherapy（RT）.However,mostly current strategies for improving RT focus only on one of the two aspects and overlook the necessity of their combinations.This study will construct a DNA dual-targeted nanodrug to simultaneously enhance the formation of DNA damage and prevent the subsequent repair,significantly enhancing the effect of radiotherapy.In addition,the nano-platform can achieve accurate RT for accurate drug delivery and real-time imaging guidance through fluorescence and magnetic resonance imaging techniques.In summary,the imaging-guided DNA dual targeting design of this study provides a new strategy for effective cancer accurate RT.Methods:1.Construction of encapsulating cisplatin prodrug and or vorinostat（globin deacetylase inhibitor）transactivator of transcription of HIV-1（TAT）and or dimethyl maleic anhydride（DA）modified PEG-b-PLGA nanomedicine(NP,NP/Pt,NP/Vor,NP/Pt+Vor and ^DANP/Pt+Vor),and characterize it,including particle size,surface potential,stability,and drug release.2.The uptake of cisplatin-loaded nanoparticles in EMT-6 cells and the level of platinumated DNA were evaluated using LSCM,ICP-MS and ultra-micro spectrophotometer.3.Acting on EMT-6 cells with Vor,NP/Vor,NP/Pt,^DANP/Vor,^DANP/Pt,NP/Pt+Vor,and ^DANP/Pt+Vor,respectively.Cellular immunofluorescence technique was used to detectγ-H2AX to evaluate DNA damage at different time points before and after radiotherapy.4.The effects of platinum-loaded nanoparticles on apoptosis were examined by flow cytometry,and the effects of voltazone nanoparticles on cell cycle and intracellular ROS levels were examined.5.To evaluate the radiosensitization effect of different drug-loaded nanoparticles by assay for colony formation.6.The EMT-6 animal model was constructed and the in vivo organ distribution,tumor enrichment and drug metabolism of nanomedicines(NP/Pt+Vor and ^DANP/Pt+Vor)were evaluated using fluorescence imaging and MRI.7.Through the tumor growth inhibition experiment,a tumor inhibition curve was drawn,the therapeutic effect of the DNA-dual-targeting nanomedicine was evaluated,and the mental state and body weight change of the experimental mice were monitored to evaluate the toxic side effects of the drug.8.The therapeutic effect of the DNA-dual-targeting nanomedicine was further studied by HE staining,TUNEL staining and Ki67 staining.Results:1.Successfully constructed related nanomedicines with suitable particle size,good stability and slow and controllable drug release capacity.2.LSCM and ICP-MS demonstrated higher plasma uptake rate and platinum-forming DNA formation rate of ^DANP/Pt,respectively.Flow cytometry experiments demonstrated that the ^DANP/Pt+Vor group significantly increased the formation ofγ-H2AX compared with the other groups over time.Different forms of vorinostat significantly increase the level of intracellular ROS.Assay for colony formation showed that the radiotherapy sensitization ratio（SER）of ^DANP/Pt+Vor was the highest.3.Fluorescence imaging and magnetic resonance imaging experiments showed that ^DANP/Pt+Vor has stronger intratumoral accumulation and retention ability,and nano drug has the highest accumulation in tumor within 24-48 hours.Tumor growth inhibition experiments demonstrated that ^DANP/Pt+Vor combined with radiotherapy has the strongest anti-tumor effect and has a tumor growth inhibition effect of nearly 90.0%.After18 days,the mice were sacrificed.HE staining of tumor tissues showed almost complete apoptosis or necrosis of tumor cells in ^DANP/Pt+Vor+RT group.TUNEL staining and Ki67 staining showed that the apoptotic rate of the tumor tissue was the highest and the proliferation rate was the lowest in this group,which further verified its anti-tumor effect.Conclutions:Successfully developed the DNA-dual-targeting nanomedicine with cisplatin prodrug and vorinostat.It has been shown to have good cellular uptake,DNA damage and radiosensitization effects at in vitro levels.The level of the body shows that the nanomedicine has obvious advantages in blood circulation and tumor enrichment,achieving synergistic effect of enhancing DNA damage and inhibiting its repair,and finally exhibits a significant anti-tumor effect.