FIN56-loaded Graphdiyne Nanoplatforms For Photothermal-ferroptosis Synergistic Therapy Against Glioblastoma Multiforme

Background: Glioblastoma multiforme(GBM)is the most malignant intracerebral tumor,accounting for 17% of primary brain tumors.About 100,000 patients diagnosed with high-grade or malignant GBM worldwide each year with a median survival of less than one year.A routine therapeutic option for GBM currently is surgical resection combined with radiotherapy and chemotherapy.Despite advances in surgical resection,radiotherapy,chemotherapy and even immunotherapy,the prognosis for GBM patients remains unfavorable,which is mainly attributed to rapid proliferation of GBM cells that leads to a recurrence after surgery,development of resistance in GBM cells to chemotherapy,and difficulty in crossing the blood brain barrier(BBB)and blood tumor barrier(BTB)for some drugs.Clinical and laboratory studies have demonstrated that monotherapy strategies often lead to resistance in GBM,resulting in a poor therapeutic response.Therefore,a combination of tumor-targeted therapy and PTT holds promising implications for clinical use in GBM treatment.Graphdiyne(GDY)is presently the only carbon-isomer containing sp-hybridized carbon atoms.Studies have reported the application of GDY in energy storage and catalysis,but it has still not been widely deployed like graphene and other materials.Research has shown that GDY,a photothermal material with drug-carrying capacity and modifiability,exerts significant antitumor effects in cancer studies by adsorbing small molecules containing aromatic hydrocarbon or aromatic hydrocarbon-like structures through π-πstacking conjugation,indicating the potential of GDY for tumor-targeted therapy and PTT therapy.In the targeting therapy,receptor-ligand recognition strategy has been widely employed.Low-density lipoprotein receptor-related protein-1(LRP-1)is a membrane protein receptor that is highly expressed in brain capillary endothelial cells,tumor neovascularization and GBM.The modification of receptor-associated protein(RAP),the ligand of LRP-1,effectively promotes drug penetration through BBB and increases the drug content in GBM tissues,indicating a favorable viability of nanocarriers modified with RAP short peptides as GBM-targeted therapeutic drugs.Ferroptosis is an iron-dependent mode of cell death characterised by oxidative damage,and glutathione peroxidase 4(GPX4)plays a critical role in mediating ferroptosis,inhibits cellular lipid peroxidation by degrading small molecule peroxides and lipid peroxides.FIN56 is a specific ferroptosis inducer which induces GPX4 degradation and results in massive production of reactive oxygen species(ROS),leading to structural damage of mitochondria and disruption of cellular energy metabolism,ultimately inhibiting tumor growth,or promoting tumor cell death.Thus,in this work,we utilize the ferroptosis property of FIN56 and the targeting property of RAP with GDY as the carrier and photothermal agent to develop a nano-drug delivery system with the ability to cross BBB and BTB,GBM targeted drug delivery,and photothermal performance.Furthermore,we have evaluated the promoting effect of the nano-drug delivery system in combination with PTT on ferroptosis in GBM cells and further discussed about the underlying molecular mechanism.Materials and methods: In this work,GDY-FIN56-RAP self-assembled nanoplatform(GFR)was prepared by a three-step method using GDY,FIN56 and RAP.In this study,transmission electron microscopy(TEM)and atomic force microscopy(AFM)were applied to characterize the nanoplatform morphology,size and thickness,dynamic light scattering(DLS)to characterize the nanoplatform hydrated particle size distribution,and infrared imager to characterize the nanoplatform photothermal properties.To evaluate GFR-induced ferroptosis in vitro,CCK8 assay was applied to detect the effect of nanoplatform on GBM cell viability,Ed U assay on GBM cell proliferation,live/dead cell staining to detect the killing effect of nanoplatform on GBM cells,Western blot to detect ferroptosis and iron metabolism-related protein levels,flow cytometry to detect cell ROS levels,and intracellular lipid peroxidation products malondialdehyde(MDA)and reduced glutathione(GSH)were measured by kits,and mitochondrial morphology was detected by TEM.Transwell and human brain microvascular endothelial cells(h BMEC)were used to construct an in vitro BTB model to detect the ability of GFR to permeate BTB.For in vivo study,4-week-old female nude mice were inoculated with luciferase-expressing LN229 cells in the right striatum to establish GBM model mice.Imaging mass spectrometry(i MScope)was applied to detect BBB and BTB penetration capacity and targeted drug delivery ability of GFR.To evaluate the anti-GBM effect of GFR in vivo,Western blot was used to detect GPX4 protein level,flow cytometry to detect ROS level of tumor tissue,TEM to detect mitochondrial morphology,kits to detect MDA and GSH content of tissue,immunofluorescence to detect 8-hydroxy-2-deoxyguanosine(8-OHd G)level and distribution,and in vivo imaging system(IVIS)was applied to monitor GBM growth.Results:(1)GDY-RAP nanosheets with a thickness of 14.5 nm,a hydrated particle size of 220 nm,and a zeta potential of ～ 15 m V.(2)With increasing FIN56/GR dosage ratio,the GFR loading content was up to 47.16 ± 3.18%,and when the dosage ratio was increased to 3,the loading content reached a plateau and showed no remarkable rise.The encapsulation efficiency decreased from 68.25±0.72 % to 18±2.28 %.(3)GFR nanoplatform is a lamellar structure with a thickness of 16.9 nm,a concentrated distribution of hydrated particle size at 255 nm,and the zeta potential was ～ 9 m V.(4)Temperature of GFR dispersion solution was significantly increased with 808 nm laser irradiation in a dose-dependent manner,and four laser switching cycles showed a favorable photothermal stability of GFR.Laser irradiation(808 nm)significantly induced GFR to release more FIN56 under acidic conditions.(5)In LN229 and T98 G cells,FIN56 decreased cell viability in a dose-dependent manner.GFR significantly reduced cell viability,and GFR combining with 808 nm laser irradiation further decreased cell viability.(6)808 nm laser irradiation promotes GFR-induced GBM cell death.(7)GFR nanoplatforms induced redox imbalance in GBM cells by decreasing GPX4 protein levels and promoting higher cellular ROS levels.(8)808 nm laser irradiation promotes GFRinduced ferroptosis in GBM cells.(9)GFR showed insignificant destruction of the in vitro BTB barrier structure over 8 h.(10)GFR effectively penetrated the in vitro BTB model and reduced the GPX4 level of GBM cells on this model.(11)i MScope results showed that the GFR nanoplatform rapidly and specifically delivered FIN56 to GBM tissues.(12)In vivo biodistribution of FIN56 demonstrated that GFR efficiently delivered FIN56 to GBM tissues and prolonged FIN56 retention time in GBM tissues.(13)GFR retained its favorable photothermal properties in vivo,and 808 nm laser irradiation induced an increase in tumor site temperature.(14)GFR nanoplatform inhibits GBM growth in combination with 808 nm laser irradiation.(15)GFR nanoplatform extends the lifespan of xenograft tumor-bearing mice in combination with 808 nm laser irradiation.(16)GFR,GR-and GFR-808 nm laser irradiation significantly improved the neurological function and locomotor ability of tumor-bearing mice.(17)GFR nanoplatform combined with NIR irradiation promotes GPX4 degradation and mitochondrial destruction in GBM tissue.(18)GFR nanoplatform combined with 808 nm laser irradiation promotes GBM ferroptosis in tumor-bearing mice,significantly inhibits GBM growth and prolongs mice’s lifespan.(19)The nanoplatforms prepared in this work resulted in neither significant hemolysis at the administered dosages nor apparent pathological lesions to the major organs of mice.Conclusion: A GBM-targeted drug delivery platform GDY-FIN56-RAP(GFR)loaded with ferroptosis inducers FIN56 was prepared for the first time in this work.Our findings demonstrate that GFR delivers FIN56 to brain and GBM cells through the specific binding of RAP with LRP-1,which leads to GBM cell death.In addition,our data illustrated the molecular mechanism by which GFR combined with PTT promotes ferroptosis by reducing intracellular GPX4 levels in GBM.The findings provide a basis not only for the potential clinical application of GFR against GBM,but also a scientific basis for further development of GDY applications in the biomedical field.