Mechanism Study Of Brain-targeted Self-assembly Nanoplatform For Enhanced Ferroptosis Therapy Of Glioblastoma

Background and Aim:Glioblastoma（GBM）is the most aggressive and lethal malignant brain tumor.Due to the existence of blood-brain barrier（BBB）,many drugs cannot reach the tumor site through BBB,resulting in the treatment of GBM is difficult to achieve the ideal effect.Notably,GBM is not sensitive to apoptosis during chemotherapy,but is sensitive to iron death.Therefore,developing treatments that can effectively target GBM and trigger iron death by crossing the BBB is critical to improving treatment outcomes.This study aims to explore the role of L-D-I/NPs in the biological function of GBM cells and the mechanism of iron death caused by targeting GBM across BBB by constructing a multifunctional biomimetic nanoplatform L-D-I/NPs.At the same time,to verify whether L-D-I/NPs can effectively inhibit the occurrence and development of in situ GBM in vivo and prolong the survival period of mouse GBM model,which lays a foundation for further exploration of the pathogenesis of GBM and provides a new idea for more effective treatment of GBM.Methods:1.Preparation and characterization of L-D-I/NPsDHA and ICG are synthesized into D-I/NPs by nano-precipitation method,and L-D-I/NPs are further modified and encapsulated using LF.Then the morphology,particle size and potential of L-D-I/NPs were detected by transmission electron microscope（TEM）and Malvern particle size potentiometer.The wavelength of the nanoparticles is detected by ultraviolet spectrogram scanning.In addition,the photothermal and photodynamic levels of L-D-I/NPs are measured using an 808 laser and 1,3-diphenylisobenzofuran（DPBF）.Finally,the release behavior of DHA and ICG from L-D-I/NPs are studied using dynamic membrane dialysis.2.In vitro cell assay of L-D-I/NPsThe uptake behavior of L-D-I/NPs are analyzed qualitatively and quantitatively by inverted fluorescence microscope.At the same time,the intracellular ROS levels are detected by DCFH-DA fluorescent probe.In addition,MTT assay analyzed the toxicity of different experimental groups to GBM cells.At the same time,the ability of L-D-I/NPs to cross BBB and kill GBM cells are evaluated by establishing in vitro BBB simulation tests.Finally,C11-BODIPY581/591 and Phen Green SK probes are used to detect lipid oxidation levels and Fe²⁺content in GBM cells,and the expression levels of ferroptosis related proteins（SLC7A11 and GPX4）are detected by Western blot.3.Animal experiments of L-D-I/NPsThe distribution of L-D-I/NPs in vivo are studied by animal imaging and the temperature changes of mouse brain tumors are recorded by infrared thermography.In addition,it is observed whether L-D-I/NPs enhanced the in vivo therapeutic properties of tumors.Finally,hematoxylin-eosin（HE）staining and immunohistochemistry are used to evaluate the morphological changes of tumor tissues.Results:1.Preparation and characterization of L-D-I/NPsThe characterization of L-D-I/NPs show that they have a structured spherical shape with a particle size of about 120nm and an electric potential of-28.2.The UV absorption pattern have a maximum UV absorption peak near 808nm.In addition,the higher the concentration of L-D-I/NPs,the better the heating effect.In addition,the photodynamic experiments show that L-D-I/NPs can produce a large amount of singlet oxygen.In PBS with p H=5.0,L-D-I/NPs released about 80%DHA within 48h.2.L-D-I/NPs promote ROS accumulation and lipid peroxidation in GBM cells,resulting in ferroptosisThe cell uptake experiment shows that L-D-I/NPs have obvious red fluorescence at 8h,which reaches the maximum uptake capacity,indicating that L-D-I/NPs have superior cell uptake behavior.The photodynamic effect shows that the fluorescence signal of L-D-I/NPs group is significantly stronger than that of other groups,indicating that the nanoparticles can produce a large number of ROS.In addition,the MTT assay shows that L-D-I/NPs exhibits the best anticancer activity compared with other groups,and also significantly better than L-D-I/NPs without laser irradiation.At the same time,we find that L-D-I/NPs can effectively cross the blood-brain barrier and kill GBM cells.The fluorescence of GBM cells changed from red to green by C11-BODIPY581/591 staining,indicating that L-D-I/NPs has the ability to cause lipid peroxidation.In addition,the green fluorescence intensity is significantly reduced in the L-D-I/NPs treatment group compared with the other groups,indicating an increase in intracellular Fe²⁺after L-D-I/NPs treatment.Western blot shows that SLC7A11 and GPX4 band signals are reduced,suggesting that L-D-I/NPs treatment may contribute to the occurrence of ferroptosis.3.L-D-I/NPs distribution and inhibition of GBM proliferation in vivoThe L-D-I/NPs group shows the strongest fluorescence signal 6h after transcaudal vein administration.In addition,the fluorescence intensity of L-D-I/NPs are the highest in the tumor sites of mice,and the surface temperature of tumor sites in the L-D-I/NPs group may be nearly 10℃higher after laser irradiation.Tumor growth is delayed and bioluminescence is lowest in L-D-I/NPs-treated mice compared to the other groups.In addition,the L-D-I/NPs group experiences relatively small weight loss,demonstrating the biosafety and therapeutic efficacy of L-D-I/NPs.Finally,HE staining shows that the L-D-I/NPs treatment group have obvious tumor damage.Meanwhile,immunohistochemical results show that the Ki67 staining is weaker in the L-D-I/NPs treatment group than in the other groups,and the expression levels of GPX4 and SLC7A11 are significantly reduced in the L-D-I/NPs treatment group.Compared with PBS group,no significant histological damage is found in the heart,liver,spleen,lung and kidney in the L-D-I/NPs group.Conclusion:1.L-D-I/NPs can bind to LRP1 and target GBM through BBB,causing ROS accumulation and lipid peroxidation in GBM cells,thus inducing ferroptosis.2.In vivo experiments have confirmed the biosafety and inhibitory effect of L-D-I/NPs on GBM.