Homologous Targeting Nanoparticles For Enhanced Pdt Against Osteosarcoma HOS Cells And The Related Molecular Mechanisms

Background:Ostesarcoma（OS）is a common primary tumor in children and adolescents.Currently,surgery and chemotherapy are mainly used in clinical treatment.However,the toxic and side effects of chemotherapy drugs and the occurrence of tumor drug resistance seriously affect the efficacy of OS,and no prominent advancements in OS treatment have been made in the past 20 years.Although photodynamic therapy（PDT）is an emerging technique for cancer therapy,the lack of targeted photosensitizers for OS treatment severely limits its applications.Purpose:To achieve the targeted therapy of OS treatment by PDT,we constructed a potential theranostic nanoplatform by using（poly（lactic-co-glycolic）acid（PLGA）nanoparticles（NPs）encapsulating IR780into the shell（PLGA-IR780 NPs）,which were further camouflaged with human OS cell membranes from the HOS cell line（MH-PLGA-IR780NPs）.Meanwhile,this study investigated the main death modes and related molecular mechanisms of MH-PLGA-IR780 NPs-mediated PDT killing OS.In view of this,this study is mainly divided into three parts:1）we constructed a potential theranostic nanoplatform by using W/O/W double emulsion and physically extruding methods,and verified the characterization of the MH-PLGA-IR780 NPs.2)These constructed NPs showed the capacity for homologous targeting with excellent photoacoustic（PA）/fluorescence（FL）imaging ability.Benefitting from their homologous targeting capacity,MH-PLGA-IR780 NPs obviously promoted cell endocytosis in vitro and tumor accumulation in vivo,which could further improve PDT performance under near-infrared（NIR）irradiation.In addition,to their homologous targeting and PA/FL dual-mode imaging ability,MH-PLGA-IR780 NPs had advantages in penetrating deeper into tumor tissues and in real-time dynamic distribution monitoring in vivo,which laid a foundation for further clinical applications in OS.3)we demonstrated that PDT guided by the constructed NPs could significantly induce HOS cells apoptosis and ferroptosis via excessive accumulation of reactive oxygen species（ROS）,and further determined that the potential anticancer molecular mechanism of apoptosis was triggered by the release of cytochrome c-activated mitochondrial apoptosis（endogenous apoptosis）,and that ferroptosis caused the activation of nuclear receptor coactivator 4（NCOA4）-mediated ferritinophagy and the inactivation of glutathione peroxidase 4（GPX4）,synergistically leading to excessive accumulation of Lipid-ROS and Lipid peroxides（LPOs）.Concurrently,MH-PLGA-IR780NPs-guided PDT also showed an obvious inhibitory effect on tumor growth in vivo.The main methods are as followings:1)Pl GA-IR780 NPs was prepared by a W/O/W double emulsion method.2)MH-Pl GA-IR780 NPs was prepared by physically extruding method.3)Ultraviolet–visible（UV–vis）,dynamic light scattering（DLS）spectrophotometer,transmission electron microscopy（TEM）were used to detect the characterization of different nanoparticles.4)The biosafety of different nanoparticles was detected by CCK-8 in vitro.5)Different nanoparticles were intravenously injected into BALB/c nude mice,and blood samples and major organs（heart,liver,spleen,lung and kidney）were harvested at 0,1,7,14,and 28days post injection.6)Homologous targeting and penetration depth of MH-PLGA-IR780 NPs were verified by confocal laser scanning microscopy（CLSM）and flow cytometry（FC）quantitative analysis in vitro.7)Homologous targeting of MH-PLGA-IR780 NPs was verified by FL imaging and PA imaging in vivo.8)ROS and ATP contents were detected by DCFH-DA probe and ATP assay kit to detect MH-PLGA-IR780 NPs guided PDT performance in vitro.9)CCK-8 assay was performed to investigate the main cell death modes.Before NIR irradiation,HOS cells were pretreated with multiple inhibitors of cell death pathways,including an apoptosis inhibitor（z-VAD-FMK）,a necrosis inhibitor（Nec-1）,an autophagy inhibitor（Baf-A1）,a ferroptosis inhibitor（Fer-1）,and a general ROS scavenger（NAC）,for 24 h.10)The apoptosis rate of HOS and the related molecular mechanism induced by MH-PLGA-IR780 NPs guided PDT were detected by JC-1,FC,Western blot assay.11)FC,Western blot and immunofluorescence（IF）were also used to detect the ability of MH-PLGA-IR780 NPs guided PDT to induce ferroptosis and its related molecular mechanism.12)The inhibitory effect of MH-PLGA-IR780 NPs guided PDT on xenograft model was verified in vivo.13)To further determine whether ferroptosis induced by targeted PDT could enhance the original killing characteristics of PDT in vivo,we administered PDT-targeted therapy to HOS xenograft tumor-bearing mice after a combination injection of MH-PLGA-IR780 NPs and DFO.14)HOS cells were preincubated with DFO and Fer-1 for 24 h followed by targeted PDT intervention to detect the relationship between apoptosis and ferroptosis in vitro by CLSM,FC,western blot assays.A list of the main results are as following:1.MH-PLGA-IR780 NPs were successfully prepared by W/O/W double emulsion and physically extruding method,and the characterization and biosafety of MH-PLGA-IR780 NPs were detected.1.1.TEM images showed the MH-PLGA-IR780 NPs were uniform in size and monodispersed,and that there was a lipid shell on the outside of the NPs wrapped by the cell membrane.The expression levels of proteins in the MH and MH-PLGA NPs groups were comparable the expression of cell membrane markers and some cellular adhesion molecules（Na⁺/K⁺-ATPase,Ncadherin,Galectin-3,Ep CAM,and Cxcr4）for source-cell-specific targeting via the homologous binding mechanism by western blot analysis.1.2.The particle sizes and zeta potentials of these NPs were measured by DLS,and the average size of MH-PLGA-IR780 NPs was distributed at approximately 236.8 nm.The zeta potential of the MH-PLGA-IR780 NPs was-10.09±0.70 m V.The UV–vis spectrum showed that the MH-PLGA-IR780 NPs had a characteristic absorption peak at 798 nm from IR780,further indicating the successful loading of IR780.1.3.To verify the safety of the MH-PLGA-IR780 NPs in vitro,a CCK-8 assay was used to examine cell viability 12 h after coincubation of a wide range of concentrations of different NPs（PLGA:0.0,0.2,0.4,0.6,0.8,1.0 mg/m L）with HOS cells,IR780-encapsulating NPs exhibited weak cytotoxicity at a higher concentration（PLGA-IR780 NPs≥0.6 mg/m L）.Then,a neutralized concentration of the NPs（PLGA:0.2 mg/m L）was used to determine the cell viability at different treatment times（0,12,24,48 h）.The CCK-8 results demonstrated that the cell viability did not change with increasing coincubation time.1.4.MH-PLGA-IR780 NPs（PLGA:5 mg/m L,200μL）were intravenously injected into BALB/c nude mice,and blood samples and major organs（heart,liver,spleen,lung and kidney）were harvested at 0,1,7,14,and 28 days post injection.The routine blood examination and serum biochemical index results showed no significant differences between the samples,and no obvious histopathological changes were observed in the aforementioned vital organs after treatment with the MH-PLGA-IR780NPs.2.Homologous targeting improves intracellular uptake and accumulation in tumors of the MH-PLGA-IR780 NPs,which can be used as tumor probe for precise tumor diagnosis and therapy.2.1.The results of CLSM and FC analysis showed homologous targeting narrows the time of intracellular endocytosis and contributes to effective phagocytosis in vitro.In vivo,FL imaging and PA imaging proved that MH-PLGA-IR780 NPs can significantly aggregate in HOS xenografts.2.2.MH-PLGA-IR780 NPs only have the potential ability to achieve tumor targeting in the HOS cell line in vitro and vivo,which had poor targeting effect on breast cancer cell 4T1,lung cancer cell A549 and other OS cell lines（MG63 and K7M2）.Moreover,MH-PLGA-IR780 NPs has no targeting effect on human osteoblast HFOB,and will not damage normal bone tissue at the same time of PDT targeted therapy.2.3.MH-PLGA-IR780 NPs has good FL intensity and PA signal in vivo,which can be used as a potential tumor probe to realize the integration of diagnosis and treatment of OS.3.Homologous targeting can significantly improve the PDT performance on OS.MH-PLGA-IR780 NPs guided PDT could significantly induce HOS cells apoptosis and ferroptosis,the potential anticancer molecular mechanism of apoptosis was triggered by the release of cytochrome c-activated mitochondrial apoptosis,and that ferroptosis caused by the activation of NCOA4-mediated ferritinophagy and the inactivation of GPX4.3.1.The results of CLSM and FC analysis showed that MH-PLGA-IR780 NPs guided PDT could produce a large number of ROS and consume intracellular ATP by DCFH-DA probe staining and ATP assay kit,further leading to mitochondrial damage.3.2.The results of western blot,CLSM and FC analysis showed that MH-PLGA-IR780 NPs guided PDT could induce HOS mitochondrial apoptosis.3.3.MH-PLGA-IR780 NPs guided PDT could produce a large number of LPOs and Lipid-ROS determined by Liperfluo and C11-BODIPY staining.The results of western blot,IF,CLSM and FC analysis demonstrated that homologous targeting-associated theranostic nanoplatform not only can decrease the activity of GPX4 by inhibiting the system Xc-transporter but also can promote the accumulation of Fe²⁺by activating NCOA4-mediated ferritinophagy to degrade ferritin（FTH and FTL）,synergistically inducing ferroptosis in HOS cells.3.4.MH-PLGA-IR780 NPs guided PDT can significantly inhibit the growth of HOS xenograft tumor with high therapeutic biosafety.3.5.Compared with the significant inhibitory effects of the targeted PDT,DFO weakened tumor growth in the xenograft model by inhibiting ferroptosis.In vitro,DFO and Fer-1 could significantly inhibit HOS apoptosis induced by MH-PLGA-IR780 NPs guided PDT.Conclusion:In summary,we successfully constructed an emerging theranostic nanoplatform（MH-PLGA-IR780 NPs）with tumor targeting ability accomplished by homologous targeting.Because of its superior FL/PA imaging performance,this nanoplatform is desirable for precise tumor diagnosis and therapy.Combining these targeting NPs with NIR irradiation synergistically induced apoptosis and ferroptosis to improve PDT efficiency in vitro and in vivo.In addition,our results suggest that the underlying mechanisms of apoptosis and ferroptosis are initiated by the mitochondrial apoptotic pathway,excessive accumulation of Fe²⁺,and inactivation of GPX4.Finally,blocking ferroptosis inhibited PDT-induced apoptosis by decreasing intracellular ROS and inhibiting the mitochondrial apoptotic pathway,suggesting that ferroptosis acts as a“death switch”in this homologous targeting-associated theranostic nanoplatform.