Dual-mode Imaging Magnetic Nanoparticles Based On Fenton Like Reaction In The Treatment Of Breast Cancer

BackgroundWhen metal ions react with hydrogen peroxide（H₂O₂）in the tumor region,the free radicals produced by Fenton like reaction can induce tumor cell death through oxidative damage mechanism,but the concentration of H₂O₂ in the tumor region can not meet the needs of tumor treatment.To solve this problem,glucose oxidase（GOx）,which can consume the accumulated glucose in tumor area,becomes a feasible scheme.In this project,we constructed a kind of magnetic nanoparticles with poly-（lactic co glycolic acid）（PLGA）as the carrier.The magnetic nanoparticles were attracted to the tumor region,and glucose was converted to H₂O₂ by GOx.Then,the magnetic nanoparticles reacted with iron ions to generate oxidative radicals.Fe₃O₄ nanoparticles,as the magnetic material,can simultaneously enhance the characteristics of PA-image/MRI and realize the integration of tumor diagnosis and treatment.ObjectiveFe₃O₄@PLGA-GOx Magnetic nano-catalyst was structured to treat tumor based on Fenten-like reaction with dual-mode imagingMethods1.The Fe₃O₄@PLGA-GOx was prepared by double emulsification.Transmission electron microscope,scanning electron microscope,Malvern laser particle size analyzer,vibrating sample magnetometer,inductively coupled plasma atomic emission spectrometry（ICP-OES）and Coomassie brilliant blue were used to characterize,measure and calculate the morphology,particle size,surface potential,hysteresis loop,drug loading rate and entrapment efficiency.2.（1）After incubating with the magnetic Fe₃O₄@PLGA-GOx nano-catalyst for 2 hours,the cells were stained with 2′,7′-dichlorodi hydrofluorescein diacetate（DCFH-DA）probe for 30 minutes.The fluorescence intensity of reactive oxygen species（ROS）was measured by Confocal laser scanning microscopy（CLSM）and flow cytometry,and the types of ROS were detected by Dihydroethidium（DHE）,Amplex-Red and Hydroxyphenyl fluorescein（HPF）.（2）The change of pH caused by the combination of different substrates and reactants in different time was monitored by pH meter to confirm the Fenton-like reaction of Fe₃O₄@PLGA-GOx magnetic nano-catalyst.In addition,through the maximum reaction rate and Michaelis constant of the Fe₃O₄@PLGA-GOx magnetic nano-catalyst for glucose/hydrogen peroxide can be obtained by using the beer Lambert law,and its activity can be confirmed.（3）1,1′-dioctadecy l-3,3,3′,3′-tetramethylcarbocyanine perchlorate（Di I）was used to label the Fe₃O₄@PLGA-GOx magnetic nano-catalyst,and the cells were,randomly divided into two groups,incubated with it.One group was attracted by magnet and the other group was the control group.After incubation,the nuclei were labeled with 4’,6-diamidino-2-phenylindole（DAPI）dye,and the relationship between the cells and the nanocatalyst was observed by CLSM.（4）Cell counting kit-8（CCK-8）cytotoxicity assay was used to detect the cytotoxicity of nano-catalyst on tumor cells.Calcein-AM and Propidium iodide（PI）were used to label living cells and dead cells respectively,and the proportion relationship between them was observed under CLSM.Meanwhile,apoptosis inhibitor APO,Nec-1,3-MA,Fer-1 and DFO were used in combination to explore the cell death mode caused by Fe₃O₄@PLGA-GOx magnetic nano catalyst3.Establish subcutaneous 4T1 breast cancer metastasis model in female BALB/c mice.When the tumor volume reached more than 50 mm³,mice were injected through the tail vein Fe₃O₄@PLGA-GOx,and in the magnetic targeting group,a permanent magnet was bound near the tumor for magnetic targeting.The distribution of nano-catalyst in vivo was observed by small animal in vivo imager to evaluate the ability of magnetic field to target tumor.After 24 hours,the in vitro fluorescence contents of main organs and tumor tissues were detected.4.By VEVO-LAZR photoacoustic imaging system and Siemens Magnetom skyra 3.0T magnetic resonance imaging system to evaluate PA-imaging/MRI-imaging ability of Fe₃O₄@PLGA-GOx magnetic nano-catalyst in vivo/vitro.（1）In vitro:different concentrations of Fe₃O₄@PLGA-GOx Magnetic nano-catalyst and pure phosphate buffer line（PBS）solution were used to image,and the relationship between concentration and photoacoustic signal/transverse relaxation rate was analyzed.（2）In vivo:female BALB/c mice with subcutaneous 4T1 breast cancer metastasis were established.In the Fe₃O₄@PLGA-GOx magnetic targeting group,a permanent magnet was bound near the tumor for magnetic targeting.The changes of photoacoustic signal and T₂ signal in tumor area were evaluated PA-imaging/MRI-imaging ability of Fe₃O₄@PLGA-GOx magnetic nano-catalyst in vivo.5.（1）Healthy female BALB/c mice were injected intravenously Fe₃O₄@PLGA-GOx.The blood glucose fluctuation of mice at different time was monitored by Roche blood glucose meter.At the same time,healthy female BALB/c mice were injected with PLGA Fe₃O₄@PLGA,PLGA-GOx and Fe₃O₄@PLGA-GOx via tail vein at different times.After taking blood from the orbit and dissecting the main organs,blood routine,serum biochemical indexes and histological analysis were carried out to evaluate its biological safety in vivo.（2）The subcutaneous 4T1 breast cancer metastasis model in female BALB/c mice were established.And the BALB/c mice were injected PLGA,Fe₃O₄@PLGA,PLGA-GOx and Fe₃O₄@PLGA-GOx into tail vein.In addition,Fe₃O₄@PLGA-GOx Group was randomly divided into with-magnetic targeting group and without-magnetic targeting group.In with-magnetic targeting group,a permanent magnet was bound near the tumor for magnetic targeting for 2 hours.The weight and tumor volume of mice were recorded every other day.After 15 days of treatment,the tumor was dissected and photographed for histological analysis.6.The 4T1 cells were incubated with PBS/Fe₃O₄@PLGA-GOx magnetic nano-catalyst for 4 hours,the cells were collected with total RNA extraction reagent,and then the transcriptome sequencing（RNA-Seq）was performed to screen out the differentially expressed genes before and after treatment,and the gene ontology was used to analyze the differentially expressed genes,The KEGG pathway was analyzed in the Kyoto Encyclopedia of genes and genomes（KEGG）.Results1.（1）Transmission electron microscope（TEM）and scanning electron microscope（SEM）images are showed the uniform spherical shape of Fe₃O₄@PLGA-GOx,and Fe₃O₄ particles were loaded on the shell.On the macro level,Fe₃O₄@PLGA-GOx because of the presence of Fe₃O₄ the color of the emulsion changed from the white PLGA emulsion to the yellowish brown emulsion.（2）The particle size of PLGA,PLGA-GOx,Fe₃O₄@PLGA and Fe₃O₄@PLGA-GOx was 222.5±36.3 nm,233.3±45.5 nm,243.7±41.0 nm and 259.5±61.6 nm respectively.The surface potential was-21.7±5.2 m V,-25.0±4.2 m V,-15.3±5.6 m V and-23.0±6.5 m V respectively.（3）By Coomassie blue method,the sealing rate and the drug loading capacity of GOx were calculate to 25.1±5.7%and 4.5±4.2%respectively.The encapsulation rate of Fe₃O₄ was 91.3±2.5%and the load was 6.3±3.2%by ICP-OES.（4）The magnetometer of vibration sample confirmed the superparamagnetism of Fe₃O₄ particles at room temperature.When Fe₃O₄was embedded in nanoparticles,the saturation magnetization decreased from37.2 emu g^-1 to 24.1 emu g^-1.In addition,Fe₃O₄@PLGA-GOx nanoparticles can gather into the external magnetic field.2.（1）The results were confirmed which the ROS of the Fe₃O₄@PLGA-GOx can be fluorescent by the generation of ROS including hydroxyl radical,superoxide anion and hydrogen peroxide by CLSM and flow cytometry.（2）The change of pH is due to the accumulation of glucolactone during the reaction of GOx,so the combination of GOx and Fe₃O₄ can accelerate the accumulation of glucolactone,which leads to a higher pH decline than that of GOx alone.In addition,the fitting of the Mies equation and the linear double reciprocal equation is used to the maximum reaction rates of Fe₃O₄@PLGA-GOx nanoparticles to H₂O₂ and glucose solutions were 7.3×10^-8 M s^-1 and 1.1×10^-8 M s^-1,and the Mier constants were 30.3 m M and15.6 m M respectively.（3）In CLSM,it can be observed that the red fluorescent nanoparticles gather around the blue nucleus,and the nanoparticles at the magnet boundary are distributed in a straight line by enlarging the field of view.It is confirmed that nanoparticles can gather around the cells and be absorbed by the cells under the action of magnetic field.CCK-8 toxicity test and CLSM can be confirmed Fe₃O₄@PLGA-GOx can effectively cause 4T1 cell death,and the death mode is a mixed mode of iron death and apoptosis.3.The fluorescence of small animals in vivo is displayed that Fe₃O₄@PLGA-GOx It can be effectively gathered in the tumor area under the action of magnetic field,and without magnetic field Fe₃O₄@PLGA-GOx Too much concentration in the spleen and liver.4.The results show that the concentration of the Fe₃O₄@PLGA-GOx particles in vitro has a linear growth relationship with the photoacoustic signal/transverse relaxation rate.In vivo experiments have confirmed that the with-magnetic target group of the photoacoustic-imaging/T2-imaging ability in the tumor area is stronger than that of the without-magnetic target group.5.（1）Although the mice are injected Fe₃O₄@PLGA-GOx The blood glucose decreased steadily in the last 30 minutes,but no pathological blood glucose was observed during the whole observation period.In the long-term observation,the mice did not show biochemical abnormality,no obvious infection,no kidney and liver damage,and hematoxylin-eosin staining（H&E）staining of the main organs（heart,liver,spleen,lung and kidney）also showed no obvious histopathological damage and the high biological safety of Fe₃O₄@PLGA-GOx.（2）During the treatment period,the weight of mice did not change significantly,while the efficacy of Fe₃O₄@PLGA and PLGA group was negligible,the growth of tumor was not significantly affected in the Fe₃O₄@PLGA group,and the inhibition rate of PLGA-GOx group was35.1%.and in the Fe₃O₄@PLGA-GOx group,the inhibition rate of the without-magnetic target group was 46.7%,and 87.4%in the with-magnetic target group.（3）The H&E staining of tumor sections showed that the tumor cells were destroyed obviously,including nuclear shrinkage,nuclear rupture and nuclear dissolution.The proliferation cell proliferation activity was evaluated by immunohistochemistry staining of proliferating cell nuclear antigen（PCNA）in tumor sections.After treatment,nanoparticles significantly inhibited the proliferation of tumor cells.In addition,the apoptosis positive cells were labeled with green fluorescence by terminal-deoxynucleididyl transfer mediated nick end labeling（TUNEL）,while nanoparticles could lead to apoptosis.6.Through RNA-Seq analysis,425 up regulated genes and 648 down regulated genes were involved in the treatment process.The go analysis analyzed the cell components,molecular functions and biological processes of these genes.In addition,KEGG pathway analysis shows that the important gene regulatory pathways involved in the occurrence,development,metastasis and prognosis of breast cancer are involved in the treatment,such as Fox O signaling pathway,IL-17 signal pathway and p53 signaling pathway.ConclusionsThis study verified the validity of the method which Fe₃O₄@PLGA-GOx can target the tumor through the external magnetic field,kill the tumor and the ability of PA-imaging/MRI dual-mode imaging.And it provides a new method for clinical treatment of tumor.