| BackgroundUveal melanoma(UM)is an aggressive ocular tumor originating from the uvea.It typically occurs in the posterior pole of the adult eyeball and can significantly impair the visual function and life health of the patients.Despite the fact that 50%of UM cases can metastasize,the risk of distant metastasis is very low for small-volume UM.Therefore,it is crucial to achieve early and timely diagnosis and intervention for UM patients,in order to reduce the possibility of distant hematogenous metastasis and ensure complete visual function and a longer lifespan.Currently,therapy for each patient is individualized.The appropriate treatment is based on the location,volume,and clinical features of the tumor as well as the basic conditions of the patients.The aim of ocular therapy for UM is to achieve local control of the tumor and reduce the risk of pathological metastasis while preserving ocular integrity and maximizing visual function.Traditional ocular treatments for UM include radiotherapy,surgery,transpupillary thermotherapy(TTT),and photodynamic therapy(PDT).External scleral plaque radiotherapy(PRT)is the most commonly used form of radiotherapy,and the prognosis of PRT is similar compared with patients undergoing enucleation.However,PRT requires skilled ocular surgeons,and the use of plaques could potentially harm healthy tissues surrounding tumors,leading to complications such as cataract,neovascular glaucoma,and retinal detachment.Resection of UM tumors could completely remove the tumor tissue while preserving the integrity of the eyeball to the greatest extent possible.Additionally,this approach enables the collection of fresh tumor specimens for future diagnosis and genetic testing.However,it’s crucial that local tumor resection is performed by an experienced ocular surgeon to minimize the risk of tumor dissemination.TTT and PDT are mainly used as alternative options for radiotherapy in the treatment of UM,and their clinical efficacy is limited,with potential complications.Therefore,exploring new ocular treatments for UM is of great significance.Chemodynamic therapy(CDT)is a new type of tumor therapy that has attracted widespread attention since it was proposed in 2016.Different from traditional cancer therapies,CDT exhibits anti-tumor effects through Fenton reaction(or Fenton-like reaction)with chemical products in the tumor microenvironment(TME).Nanomaterials composed of transition metals,such as divalent iron ions(Fe2+),have the ability to react with hydrogen peroxide(H2O2)present in TME to generate hydroxyl radicals(·OH),which possess strong oxidizing properties.This reaction could result in lipid peroxidation and protein inactivation of tumor cells,eventually lead to cell death.Importantly,in normal tissues,the concentration of H2O2 is low,and therefor nanomaterials could not produce enough·OH to cause significant damage.This property makes these nanomaterials ideal for tumor-specific therapy of UM.However,traditional transition metals show limited catalytic efficiency and low degradability in vivo.The potential long-term toxic effects of these nanomaterials need to be concerned.Therefore,it is necessary to develop new CDT nanomaterials with efficient Fenton reactivity and high biosafety for the ocular treatment of UM.In our previous study,we successfully prepared non-oxidized MXene-Ti3C2Tx quantum dots(NMQDs-Ti3C2Tx)through a micro-explosion method designed by ourselves.NMQDs-Ti3C2Tx has an ultra-small size.The average transverse diameter of NMQDs-Ti3C2Tx is about 7.23nm and the average thickness is about 5nm.NMQDs-Ti3C2Tx shows stronger Fenton-like catalytic performance compared with Fe2+.NMQDs-Ti3C2Tx has a killing effect on breast and cervical cancer cells and shows excellent biocompatibility.However,the effect of NMQDs-Ti3C2Tx on UM cells and the intraocular biocompatibility of NMQDs-Ti3C2Tx are unknown and have not been reported.In this study,we explore the effect of NMQDs-Ti3C2Tx on biological behaviors of UM cells such as proliferation,invasion and migration.We also investigate the molecular mechanism of NMQDs-Ti3C2Tx on UM cells.Additionally,we evaluate the toxicity of NMQDs-Ti3C2Tx on intraocular cells and assess the intraocular biocompatibility of NMQDs-Ti3C2Tx.Part I the effect and the molecular mechanism of NMQDs-Ti3C2Tx on the biological behaviors of UM cellsObjectiveBiological behaviors of tumor cells such as proliferation,invasion and migration are closely related to tumorigenesis and metastasis.This section aims to investigate the effect of NMQDs-Ti3C2Tx on UM cell biological behaviors and further explore the mechanism of NMQDs-Ti3C2Tx on UM cells.Methods1.The preparation of NMQDx-Ti3C2TxFirst,we synthesized NMQDs-Ti3C2Tx,the raw material of NMQDs-Ti3C2Tx by traditional etching,and then characterized MXene-Ti3C2Tx by the scanning electron microscopy(SEM).After that,we used the micro-explosion method to prepare NMQDs-Ti3C2Tx and characterized NMQDs-Ti3C2Tx using the transmission electron microscope(TEM).2.Cell culture and treatmentHuman UM cell line C918,and UM cell line MUM-2B were cultured in RPMI-1640 with or without 10%fetal bovine serum(FBS).All the cell lines were grown in a 37℃incubator with 5%CO2.The main treatment is as follows:To analyze the effect of NMQDs-Ti3C2Tx on the cellular processes of UM cells,the prepared NMQDs-Ti3C2Tx was co-cultured with UM cells and the UM cell behaviors were observed and analyzed.3.UM cell viability assayCell counting kit-8(CCK-8)assay was used to detect the UM cell viability.C918 and MUM-2B cells were cultured with different concentrations of NMQDs-Ti3C2Tx(0μg/mL,25μg/mL,50μg/mL,100μg/mL,150μg/mL and 200μg/mL),respectively.After 12 hours,24 hours,and 36 hours,the absorbance was measured by using a microplate reader.4.Clone formationC918 and MUM-2B cells were cultured with different concentrations of NMQDs-Ti3C2Tx,as consistent with the CCK-8 assay,and the solution was changed every 3 days.After 14 days,the UM cells were collected to count the number of clones and the cloning rate was calculated.5.Living/Dead cell stainingC918 and MUM-2B cells were cultured with different concentrations of NMQDs-Ti3C2Tx.After 6 hours,live/dead cell staining was performed to observe the cell viability and mortality of UM cells.The results,along with the CCK-8 assay results,were further used to determine the appropriate experimental concentrations and stimulation times for subsequent experiments.6.Wound-Healing Test and Transwell TestC918 and MUM-2B cells were stimulated with different concentrations of NMQDs-Ti3C2Tx for 6 hours.After then,the cells were collected for scratch experiment and Transwell test to observe the effect of NMQDs-Ti3C2Tx on the migration and invasion ability of UM cells.8.The detection of reactive oxygen species(ROS)and lipid peroxidation in UM cells.C918 cells and MUM-2B cells were cultured with different concentrations of NMQDs-Ti3C2Tx for 6 hours.Then,cells were stained using the ROS detection fluorescent probe(H2DCFDA)and the lipid peroxidation detection fluorescent probe(C11 BODIPY 581/591).Flow cytometry analysis was final performed.9.The detection of malondialdehyde(MDA)in UM cells.After being stimulated with different concentrations of NMQDs-Ti3C2Tx for 6 hours,Cell Lysis Buffer was used to extract the total protein in UM cells.A BCA kit was used to determine the protein concentration.Then,the MDA kit was used to determine the MDA level.10.The detection of glutathione(GSH)in UM cells.After being stimulated with different concentrations of NMQDs-Ti3C2Tx for 6 hours,a GSH kit was used to determine the GSH levels in UM cells.11.The detection of membrane potential(MMP)in UM cells.After being cultured with different concentrations of NMQDs-Ti3C2Tx for 6 hours,C918 cells and MUM-2B cells were stained using the fluorescent probe(TMRM).Flow cytometry analysis was final performed.12.The observation of mitochondrial and lysosomal morphology in UM cells.After being cultured with different concentrations of NMQDs-Ti3C2Tx for 6 hours,C918 cells and MUM-2B cells were fixed using fixation solution.Mitochondrial and lysosomal morphology were then observed using a transmission electron microscopy.13.The colocalization between NMQDs-Ti3C2Tx and organelles in UM cells.To label mitochondria and lysosomes in UM cells,we used Mito-Tracker Red and Lyso-Tracker Red,respectively.A fluorescence microscope was used to observe the colocalization between NMQDs-Ti3C2Tx and organelles in UM cells.14.qPCRTotal RNA was extracted using a total RNA kit,and a reverse transcription kit was used to reversely transcribe the mRNA.Then,the Ct values of SLC7A11、PTGS2 and LC3 were obtained by qPCR.With GAPDH as the reference gene,the obtained Ct values were calculated.15.Data statistics and analysisAll experiments were conducted at least 3 times,and the data were analyzed using the software GraphPad Prism 7.A p-value of less than 0.05 is statistically significant.Results1.NMQDs-Ti3C2Tx was successfully prepared.We used electron scanning electron microscopy to characterize MXene-Ti3C2Tx,and we found that MXene-Ti3C2Tx has the "accordion"-like structure of typical MXene.The NMQDs-Ti3C2Tx was characterized by transmission electron microscopy,and we found that NMQDs-Ti3C2Tx had a regular disk-like shape.These results indicated that NMQDs-Ti3C2Tx was successfully prepared.2.NMQDs-Ti3C2Tx inhibited the proliferation and clone formation of UM cells.The CCK-8 results indicated that the proliferation and clone formation of C918 and MUM-2B cells was significantly inhibited after co-culture with NMQDs-Ti3C2Tx,and NMQDs-Ti3C2Tx played the significant inhibitory effect in a concentration-dependent manner.Live/dead staining showed that after 6 hours of stimulation with NMQDs-Ti3C2Tx(50μg/mL and 100μg/mL),a small number of dead cells appeared in C918 and MUM-2B cells,while after 6 hours of stimulation with NMQDs-Ti3C2Tx using 150μg/mL,a large number of dead cells appeared in UM cells.In the subsequent experiments,in order to avoid the influence of cell death on the experimental results,50μg/mL and 100μg/mL of NMQDs-Ti3C2Tx were selected as the experimental concentration,and 6 hours were used as the stimulation time.3.NMQDs-Ti3bC2Tx inhibited the migration and invasion of UM cells.UM cells were stimulated with NMQDs-Ti3C2Tx(0μg/mL,50μg/mL and 100μg/mL)for 6 hours.After then,wound-healing and Transwell assays were performed.The results showed that NMQDs-Ti3C2Tx significantly inhibited the invasion and migration ability of C918 and MUM-2B cells compared to the control group(0μg/mL).4.NMQDs-Ti3C2Tx disrupted the redox homeostasis in UM cells.We found that after being stimulated with NMQDs-Ti3C2Tx for 6 hours,the total ROS level and the degree of lipid peroxidation in UM cells increased compared to the control group(0μg/mL)·In addition,the GSH contents also significantly decreased.These results indicated a disturbance in the redox homeostasis of UM cells.5.NMQDs-Ti3C2Tx caused ferroptosis in UM cells.The qPCR results demonstrated that the mRNA levels of SLC7A11 and PTGS2 genes in C918 and MUM-2B cells were upregulated after being cultured with NMQDs-Ti3C2Tx.In addition,the mitochondria cristae in UM cells decreased and the mitochondrial membrane was ruptured.These indicated that the occurrence of ferroptosis in UM cells after stimulation with NMQDs-Ti3C2Tx.6.NMQDs-Ti3C2Tx caused mitochondrial homeostasis disruption in UM cells.Using Mito-Tracker Red,we stained UM cells and observed colocalization of NMQDs-Ti3C2Tx with mitochondria,indicating a potential direct attack on UM cell mitochondria.Further analysis of MMP in UM cells revealed a significant reduction after stimulation with NMQDs-Ti3C2Tx,indicating mitochondrial damage.We further used transmission electron microscopy to directly observe mitochondrial morphology,and we found that mitochondrial morphology changed significantly,indicating that NMQDs-Ti3C2Tx caused disruption of mitochondrial homeostasis.7.NMQDs-Ti3C2Tx caused lysosomal destruction in UM cells.The qPCR results revealed an up-regulation of the autophagy-related LC3 gene mRNA levels in UM cells.Additionally,transmission electron microscopy showed damaged mitochondria in autophagic lysosomes,indicating that mitochondrial autophagy occurred after stimulation with NMQDs-Ti3C2Tx.We observed black-gray particles in autolysosomes and a disruption of autophagic lysosomal membrane integrity.Furthermore,the colocalization of NMQDs-Ti3C2Tx with lysosomes in UM cells suggested that NMQDs-Ti3C2Tx was metabolized in lysosomes,and the black-gray particles in autolysosomes were NMQDs-Ti3C2Tx engulfed by lysosomes.NMQDs-Ti3C2Tx further reacted in the acidic lysosomal environment,leading to lysosomal destruction.Conclusion1.NMQDs-Ti3C2Tx could significantly inhibit the biological behaviors,including proliferation,clone formation,invasion and migration of UM cells(C918 and MUM-2B);2.NMQDs-Ti3C2Tx caused ferroptosis in UM cells by disrupting the redox balance;3.NMQDs-Ti3C2Tx attacked UM cell mitochondria,resulting in mitochondrial damage and triggering mitophagy;4.NMQDs-Ti3C2Tx disrupted lysosomes,which leads to the termination of mitophagy and exacerbates cell death.Part Ⅱ the effect of NMQDs-Ti3C2Tx on UM in nude mice and the biocompatibility of NMQDs-Ti3C2Tx in the eyeballObjectiveIn part I,we observed in vitro that NMQDs-Ti3C2Tx inhibited the biological behaviors of UM cells such as proliferation,clone formation,invasion and migration by disrupting redox metabolism,mitochondrial homeostasis,and other mechanisms.However,the impact of NMQDs-Ti3C2Tx on UM tumors in vivo remains unclear.In this section,the effect of NMQDs-Ti3C2Tx on UM was examined by constructing a nude mouse tumor model.Furthermore,the effect of NMQDs-Ti3C2Tx on intraocular cells were observed by culturing with retinal pigment epithelium(RPE)cell,and the biocompatibility of NMQDs-Ti3C2Tx in the eye was assessed through intraocular injection.Methods1.Experimental animals:UM subcutaneous tumor model:BALB/c nude mice;Intraocular injection model:C57BL/6 mice.2.Animal experiments grouping:Eight 4-week-old female BALB/c nude mice were selected and subcutaneous UM tumors were established by injecting C918 cells.The mice were then randomly divided into two groups(n=4)for drug treatment,with injections given every 2 days:a control group(treated with normal saline)and a group treated with NMQDs-Ti3C2Tx.Six 4-week-old female C57BL/6 mice were randomly divided into two groups,and 3 mice in each group:control group(normal saline)and NMQDs-Ti3C2Tx group.3.Cell culture and treatmentIn this experiment,C918 cell line was cultured as in the first part;Human RPE cell line ARPE-19 was cultured in DMEM with 10%FBS,and the cell line was grown at 37℃ and 5%CO2.The main treatment is as follows:The prepared NMQDs-Ti3C2Tx was co-cultured with RPE cells to analyze and observe the effect of NMQDs-Ti3C2Tx on the biological behaviors of RPE cells.After 6 hours of stimulation with NMQDs-Ti3C2Tx,flow cytometry analysis was performed to observe the change of ROS,lipid peroxide and MMP in RPE cells.4.Body weight and tumor volume of miceThe weight of nude mice was measured 3 times using an electronic balance before each injection,and the average of the measurements was taken as the final weight data.The length and diameter of the tumor were measured using a vernier caliper,and the tumor volume was calculated based on the results.5.The intravitreal injection in C57BL/6 miceAfter anesthesia of C57BL/6 mice,surface anesthetic was added and the pupil was dilated.After then,an intravitreal injection was administered using a Hamilton 33G puncture needle Three days after surgery,mice were subjected to fundus optical coherence tomography(OCT).6.Tissue sampling of miceAt the end of the experiment,nude mouse tumors and C57BL/6 mouse eyeballs were retained.Part of the tissues were put into liquid nitrogen for freezing,and then transferred to-80℃ for storage,while some tissues were fixed with 4%paraformaldehyde(PFA)for subsequent experiments.7.Histopathological testingTissue sections were prepared for nude mouse tumors and C57BL/6 mouse eyeballs,and then Hematoxylin-eosin(H&E)staining was performed.8.Data statistics and analysisAll experiments were conducted at least 3 times,and the data were analyzed using the software GraphPad Prism 7.A p-value of less than 0.05 is statistically significant.Results1.NMQDs-Ti3C2Tx inhibited the proliferation of UM subcutaneous tumors.Through the subcutaneous tumor experiment in vivo,we found that the volume and weight of the tumor body in the NMQDs-Ti3C2Tx group were significantly smaller than that in control group.In addition,we found that a large area of necrosis appeared near the injection site of NMQDs-Ti3C2Tx,while the tumor tissue far from the injection site was not affected.2.NMQDs-Ti3C2Tx had no effect on the viability of RPE cells.Through CCK-8 experiments,we found that even after being stimulated for 36 hours with 200μg/mL NMQDs-Ti3C2Tx,the viability of PRE cells was not significantly affected.In live dead staining experiments,we also did not observe significant cell death.3.NMQDs-Ti3C2Tx had no effect on the redox metabolism and mitochondrial homeostasis in RPE cells.The total ROS and lipid peroxide levels in RPE cells did not increase when stimulated with NMQDs-Ti3C2Tx.We also observed that there was no significant change in MMP in RPE cells after being stimulated with NMQDs-Ti3C2Tx.4.NMQDs-Ti3C2Tx had no significant destructive effect on intraocular tissues of mice.After intraocular injection of NMQDs-Ti3C2Tx,we observed through OCT that there was no significant difference in fundus images between NMQDs-Ti3C2Tx group and control group,and no significant changes in retinal thickness.Further,we found by H&E staining that the cell morphology of the eye slices from both groups was regular and there was no significant difference.Conclusion1.NMQDs-Ti3C2Tx demonstrated a powerful antitumor effect by significantly inhibiting the growth of UM tumors at the injection site in nude mice.2.NMQDs-Ti3C2Tx exhibited no toxic effects on RPE cells and demonstrated good biocompatibility within the eye. |
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