Glutathione Responsive Nanoparticles Co-loaded With Doxorubicin Prodrug/?-Lapachone Combined With HIFU For Breast Tumor Therapy

BackgroundHigh intensity focused ultrasound(HIFU),as a new technology for the treatment of benign and malignant solid tumors,is widely applied in clinical due to its non-invasive advantages.However,there are still some problems such as long treatment time,high treatment power and unsatisfactory treatment efficiency.For these problems,the nanoparticles focuses on synergies,combination therapy and visualization,continuously optimizes and innovates,plays a great role in improving the effect of HIFU on tumor.In this study,we constructed a GSH responsive nanoparticles(P@BDOX/?-Lapachone-NO-NPs)to remodel acoustic environment of tissue,increase the cavitation effect,and enhance the efficacy of HIFU ablation,improve tumor therapeutic effect.ObjectiveTo explore P@BDOX/?-Lapachone-NO-NPs synergistic HIFU ablation to improve the efficacy of tumor treatment,which is expected to provide an idea for clinical synergistic treatment of HIFU tumor.Methods1. P@BDOX/?-Lapachone-NO-NPs were prepared by esterification reaction,nitrification reaction and nanoprecipitation method,and its chemical structure,particle size,Zeta potential,morphological characteristics,drug loading capacity,in vitro stability,drug release and the NO production ability were tested.2.(1)CCK-8 cytotoxicity assay was used to detect the synergistic effect of DOX and?-Lapachone of different proportions,and to determine the optimal synergistic ratio;To detect the toxicity of nanoparticles to normal HUVECs and MDA-MB-231 breast cancer cells.(2)Cells were incubated with different concentrations of P@NO-NPs,and the content of NO was determined in cell culture medium at different time points.Cells were incubated with different formulations of nanoparticles for 4h,and then incubated with DCFH-DA probe for 30 min,cells were collected,the fluorescence intensity of ROS and DOX was determined by flow cytometry.(3)After the cells were incubated with different formulations of nanoparticles for 4 h,the cells were incubated with DAF-FM-DA and DCFH-DA probes for 30 min,respectively.The nuclei were stained with Hoechst 33342,and the uptake and distribution of DOX and the production of ROS and NO in the cells were observed by CLSM.3.(1)Female SD rats were injected with free DOX,BDOX/?-Lapachone-NO-NPs,P@BDOX/?-Lapachone-NO-NPs in the tail vein,and blood sample was taken from the orbit at different time points.After the blood samples were processed,and the concentration of DOX/BDOX was measured with UV-vis to detect the plasma pharmacokinetics.(2)Blood was taken from the orbit of healthy female BALB/c mice.After red blood cells were extracted,incubated with different concentrations of P@BDOX/?-Lapachone-NO-NPs for 4 h.The hemolysis rate of red blood cells was observed to detect the biological safety in vivo.(3)To establish a model of subcutaneous breast cancer xenograft in nude mice.When the tumor volume reached 100 mm~3,free DOX,BDOX/?-Lapachone-NO-NPs and P@BDOX/?-Lapachone-NO-NPs were injected into the tail vein.15min and 12 h after injection,the tumor site was irradiated with low intensity focused ultrasound.The drug distribution and tumor targeting accumulation ability in vivo were observed with the IVIS imaging system at different time points.The nude mice were sacrificed after 24 h,and the main organs and tumors were taken for ex-vitro fluorescence imaging.Then the main organs and tumors were ground,and the concentration of DOX was detected by UV-vis after the drug was extracted.The nude mice were injected with free DOX,BDOX/?-Lapachone-NO-NPs,P@BDOX/?-Lapachone-NO-NPs.After 24 h,DAF-FM-DA and DCFH-DA probes were injected intratumorally.1.5 h after injection,nude mice were sacrificed,tumors were dissected,frozen sections were stained with DAPI,and ROS/NO generation and the distribution of DOX in the organization were observed using CLSM.4.(1)To establish a model of subcutaneous breast cancer xenograft in nude mice.When the tumor diameter reached 1 cm,saline and P@BDOX/?-Lapachone-NO-NPs were injected into the tail vein.15 min and 12 h after injection,the tumor site was irradiated with low intensity focused ultrasound.After 24 h,the tumor point ablation was performed by JC-200 focused ultrasound tumor treatment system with irradiation power of 150 W and irradiation time of 5 s,and the gray changes of tumor tissues were observed.Take tumor tissue to measure ablation volume,calculate energy efficiency factor(EEF),and observe pathological morphology.tumor-bearing mice were randomly divided into 8 groups:?saline,?free DOX,?BDOX/NO-NPs,?BDOX/?-Lapachone-NO-NPs,?P@BDOX/?-Lapachone-NO-NPs,?US+P@BDOX/?-Lapachone-NO-NPs,?HIFU+saline,?HIFU+P@BDOX/?-Lapachone-NO-NPs.Tumor growth and survival time were observed after treatment.After 25 days,one of the nude mice was sacrificed.The main organs and tumor tissues were stained for HE.(3)Another group of mice were also treated for blood routine and blood biochemical analysis after 25 days of treatment to evaluate the in vivo biological safety of the nanoparticles.Results1.(1)~1H NMR and FTIR showed that the Mal-PEG-NO-Glu A carrier material was successfully synthesized.(2)The average particle size of P@BDOX/?-Lapachone-NO-NPs was 118.7±7.2 nm(PDI=0.231±0.02),and the Zeta potential was 1.83±0.6 m V.(3)TEM showed that P@BDOX/?-Lapachone-NO-NPs were spherical,regular in shape and uniform in size.The drug loading capacity was 10.54%.(4)The BDOX release details of P@BDOX/?-Lapachone-NO-NPs was measured in PBS and 10 m M GSH PBS solution for 48 h.The results showed that at 48 h,the BDOX release rate reached 82.87%in the presence of 10 m M GSH,and only 22.01%in PBS solution,suggesting that P@BDOX/?-Lapachone-NO-NPs was reduction-sensitive.(5)In vitro NO production test results showed that with the increase of P@NO-NPs and GSH concentrations,the production of NO increased.2.(1)The cytotoxicity of MDA-MB-231 breast cancer cells showed that the best synergistic effect was found when the ratio of DOX to?-Lapachone was 8:2.Compared with the control group,P@BDOX/?-Lapachone-NO-NPs group had the best killing effect on tumor cells.The nanoparticles has no cytotoxicity to human normal HUVECs cells,considering with excellent biosafety.(2)The results of NO generation test of MDA-MB-231 cells in vitro showed that with the increase of P@NO-NPs concentration and incubation time,the amount of NO production also increases.Flow cytometry results showed that abundant ROS were generated by nanoparticles after co-incubation with MDA-MB-231 cells.(3)CLSM show that the nanoparticles can be effectively taken up by tumor cells.Compared with the control group,the P@BDOX/?-Lapachone-NO-NPs group had stronger nuclear fluorescence and ROS fluorescence,suggesting P@BDOX/?-Lapachone-NO-NPs are effectively internalized into tumor cells.The CLSM of P@NO-NPs showed that the green fluorescence(NO)was distributed in the cells,and with the increase of concentration,the green fluorescence is stronger.3.(1)BDOX-NO-NPs and P@BDOX/?-Lapachone-NO-NPs with good blood circulation stability,and enhance the EPR effect of the nanoparticles.(2)It is resulted that RBCs can remain the structural integrity with minimal hemolysis(hemolysis rate<5%)in the presence of P@BDOX/?-Lapachone-NO-NPs at a high concentration up to 200?g/m L during the whole observation period.These P@BDOX/?-Lapachone-NO-NPs were postulated to be biocompatible.(3)In vivo imaging and tissue distribution indicate that P@BDOX/?-Lapachone-NO-NPs have better ability to target tumor accumulation than other treatment groups.results of CLSM imaging showed that after the nanoparticles accumulated at the tumor site and internalized into the cells,producing abundant ROS and NO.The P@BDOX/?-Lapachone-NO-NPs treatment group,it can be observed that DOX is distributed throughout the cell and enters the nucleus.4. P@BDOX/?-Lapachone-NO-NPs can significantly enhance HIFU ablation,and the gray difference changes significantly after ablation.The ablation volume of tumor tissue is significantly larger than that of saline group,and the energy efficiency factor is smaller than that of saline group.HIFU combined with P@BDOX/?-Lapachone-NO-NPs effectively inhibited the proliferation of tumor cells(IRT=99.7%),promoted apoptosis,and significantly prolonged the survival time of tumor-bearing mice.The results of blood biochemical and blood routine analysis showed that P@BDOX/?-Lapachone-NO-NPs had good biological safety and avoided the damage of free DOX to main tissues and organs.ConclusionsThis study verified that P@BDOX/?-Lapachone-NO-NPs can enhance the ablation of HIFU,providing a new synergist for HIFU in the treatment of tumors;HIFU combined with chemotherapy can better treat tumors,and provide a new idea for clinical treatment of tumors.