Toxicity Of Mesoporous Silica Nanoparticles Loaded With Silver Ions To The Cornea And Its Mechanisms

Background and purpose:The new environmental pollution and occupational exposure issues were caused by the mass production and widespread application of nanomaterials.Nanomaterials are widely used in cosmetics,food packaging,therapeutics and biosensors,leading to extensive exposure of the human to nano-material-rich environments with potential toxic effects [1-3].Therefore,during the past decade,more and more studies have focused on the toxicity of nanomaterials to the lung,skin,liver,kidney and spleen [4-6].However,eyes,as a superficial organ,are often neglected in the evaluation of toxicity of nanomaterials.Compared with other internal organs,eyes usually directly interact with nanomaterials may cause greater toxicity.Although the large-size particles can be blocked from the ocular surface through blinking and tear film,the small-sized nanoparticles adhere to the cornea for a long time after contacting the ocular surface,and can cross the ocular surface barrier to reach the posterior segment of the eye.Continuous exposure to environmental pollutants,such as dust,diesel exhaust particles and nano materials,may cause adverse effects on the eyes [7-8],induce serious eye diseases and even blindness [9-10].Therefore,the safety evaluation of nanomaterials to eyes has has attracted widespread attention,worldwide but but it is still in the preliminary stage.So far,research on the toxicity of nanoparticles to the eyes has focused on the effects of different NPs types,NPs size,surface charge,oxidation state,and exposure time [11-13].However,to our best knowledge,the ocular toxicity effect of another important NPs feature,large surface area,hasn't been investigated.It is well known that the interaction between NPs and biosystems plays a key role in its toxicological effects [14-16].The large surface area provides a great proportion of their atoms to be exposed on the surface [17],which makes NPs easily attach with surrounding chemical or biological molecules,and thus further enhances its surface reactivity and toxic effects [18].For instance,studies have shown that an increased surface area of NPs is considered to be efficient to initiate inflammatory response [19].Moreover,porous NPs with large surface area is reported to exert more systemic toxicity such as vasculature damage caused by porous NPs-protein interaction when compared with nonporous NPs [20].Therefore,the typical feature of nanomaterials with large surface area may also play an important role in affecting its toxicity profile to eyes.The typical feature of nanomaterials with large surface area needs to be considered when measuring the biological toxicity of NPs.Recently,silica nanoparticles(SiNPs)have been mass-produced and widely used due to their easy-to-adjust scales and morphologies,and are one of the world's highestyielding nanomaterials [21].There are two forms of SiNPs,non-mesoporous SiNPs(NSiNPs)and mesoporous SiNPs(MSiNPs).The porosity of MSiNPs endows them can be used as a functional carrier for drug transport,such as intraocular administration,oral administration drug and anti-cancer drug delivery [22-23].However,the large surface of MSiNPs may also increase its toxic effect because of its high reactivity and strong absorption capacity.Many studies have reported that MSiNPs exposure could induce oxidative stress and DNA damage,apoptosis and inhibit cell proliferation [26-27].In addition,studies have reported that MSiNPs can cause liver damage when injected into the body through intravenous injection [28].Ophthalmic drug delivery is an interesting area in current research.Silica nanoparticles(SiNPs)are promising drug carriers for ophthalmic drug delivery.It is known that corneal blindness associated with angiogenesis includes corneal diseases caused by pathological angiogenesis,resulting in visual impairment.Studies have found that MSiNPs have an anti-angiogenic effect in inhibiting corneal neovascularization caused by chemical burns [22].Another study showed that MSiNPs are used for anterior glaucoma drug delivery therapy [23].Thus,the cornea is frequently contact with MSiNPs.However,its toxic effect on the cornea has not been reported.This study will investigate the toxic effects of MSiNPs,which with large surface area,on the cornea.In addition,other toxic substances are easily adsorbed by MSiNPs,leading additional harmful effects and produce synergistic toxicity [29-30].Since silver products are widely used in daily life and medical hygiene [31-33],silver products can continuously release silver ions,and Ag~+ exhibits high cytotoxicity,which could lead to significant reduction in cell viability,DNA damage,and genotoxicity [34-37].Ag~+ exposure to air usually coexists with various pollutants including NPs Adsorption by MSiNPs may greatly enhance the uptake of Ag~+ by cells and accumulation within cells,further causing servere cytotoxicity [38-39].In this study,Ag~+ combined with MSiNPs was applied to investigate the toxic effects of NPs on Cornea.Materials and Methods: Part I: Synthesis and characterization of NSiNPs,MSiNPs and MSiNPs-Ag~+·The morphology and size of the synthesized FITC-MSiNPs and FITC-NSiNPs were characterized by a scanning electron microscope(S-4800,Hitachi,Japan)and transmission electron microscope(TEM,JEM 2100 Plus).·The hydrodynamic size of FITC-MSiNPs and FITC-NSiNPs were determined by dynamic light scattering(DLS,Omni).·The zeta potentials of FITC-NSiNPs and FITC-MSiNPs suspended in aqueous solution were measured by a Nano Brook Zeta Plus(Brookhaven,New York,NY,USA).·FITC-NSiNPs and FITC-MSiNPs were dissolved in PBS to observe the dispersion of the solution for how long.The green fluorescence of the synthesized FITC-MSiNPs and FITC-NSiNPs were detected under ultraviolet illumination(365 nm wavelength).·Surface area of FITC-MSiNPs were determined by BET equation method.Pore size distribution curve was computed by BJH method and defined as the maximum of the pore size distribution curve.·MSiNPs-Ag~+ was prepared by cyclic adsorption of heavy metal Ag~+Part II: The cytotoxicity and mechanism of NSiNPs,MSiNPs and MSiNPs-Ag~+ exposure on hCECs·Culture and identification of human corneal epithelial cells(hCECs).? ·The Cell Counting Kit-8(CCK-8)test was used to detect the effects of different concentrations of NSiNPs,MSiNPs,and MSiNPs-Ag~+ on the cell viability of hCECs.·Flow cytometry was used to detect the effect of MSiNPs and MSiNPs-Ag~+ on apoptosis of hCECs.·Immunohistochemical analysis was applied to investigate the effects of MSiNPs and MSiNPs-Ag~+ treatment on ROS levels and DNA damage in hCECs.·Transmission electron microscopy was used to analyze the effects of MSiNPs and MSiNPs-Ag~+ on the subcellular structure and mitochondrial structure of hCECs.·RNA sequencing(RNA-seq)analyzed the effects of MSiNPs and MSiNPs-Ag~+ on the transcriptome of hCECs,and identified the core pathways of the toxic effects of MSiNPs-Ag~+ on hCECs.Part III: Ocular surface damage and dry eye symptoms when exposed to MSiNPs and MSiNPs-Ag~+ and the therapeutic effects of FBS·Distinguish the adsorption capacity of MSiNPs and NSiNPs via the adsorption experiment with rhodamine B(Rh B)as the model dyes.·FBS was used as an adsorption reagent to form protein corona around NPs.The ability of MSiNPs and NSiNPs to form protein corona was detected and distinguished by adsorption experiments.·Investigated the damage effects of MSiNPs and MSiNPs-Ag~+ at the concentration of 100 ?g/m L exposure to rats corneal(in vivo)through one-week repeated exposure study.The right eyes of each group were treated with the MSiNPs and MSiNPs-Ag~+ three times-daily,at six-hour intervals,for consecutive one week.The other eye was dropped with PBS and served as the control.·The fluorescein staining test was carried out one week after exposure.3% fluorescein was dropped into the saccus conjunctivae of rats.Anterior section OCT was used to evaluate the difference of corneal injury between MSiNPs and MSiNPs-Ag~+ treatment.·After continuous exposure for one week,the apoptosis cells in the rat treatment with MSiNPs and MSiNPs-Ag~+ was labeled with TUNEL staining with whole-mount corneal immunofluorescence.·100% FBS was applied to the right eye of rats 5 min every time after exposure to MSiNPs and MSiNPs-Ag~+,The method of detecting the therapeutic effect of FBS on MSiNPs and MSiNPs-Ag~+ exposed to hCECs-induced cytotoxicity was the same as that in 2,3Results:Patr? Synthesis and characterization of NSiNPs,MSiNPs and MSiNPs-Ag~+·NSiNPs and MSiNPs were obtained through synthesis,and MSiNPs-Ag~+were obtained through heavy met al adsorption.Scanning electron microscopy(SEM)found that the average size distributions of NSiNPs and MSiNPs under were measured as~80nm.The hydrodynamic diameters in water were ~80 nm and ~86.3nm,respectively.The Zeta potentials of NSiNPs and MSiNPs were-57.61 mV and-32.17 mV,respectively.Obviously,pores were identified under TEM from MSiNPs.Bright green fluorescence under UV irradiation also demonstrated that FITC was doped in the matrix of NSiNPs and MSiNPs successfully.In addition,NSiNPs and MSiNPs were found to be well dispersed in Phosphate Buffer Saline(PBS)without coagulation.·The typical nitrogen adsorption-desorption isotherms showed specific surface areas of FITC-MSiNPs is 238.9 m2/g using Brunauer,Emmett and Teller method(BET)equation method,Besides,the total pore volume of FITC-MSiNPs was determined as 0.25 cm3/g by the Barrett-Joyner-Halenda method(BJH)equation methodt.Part II:Tne cytotoxicity and mechanism of NSiNPs,MSiNPs and MSiNPs-Ag~+exposure on hCECs·The hCECs were isolated from human corneal tissues,with tight junctions and a pebble-like structure.The positive rates of corneal epithelial cell-specific markers CK12 and CK15 are 100%,in dicating isolated pure hCECs.·The increased cell death of hCECs was combined with the increase of NPs concentration confirmed by Phase contrast microscope observation.Both 100 ?g/mL MSiNPs and MSiNPs-Ag~+could cause hCECs cell death,and the number of dead cells caused by MSiNPs-Ag~+was significantly higher at the same concentration of MSiNPs.Flow cytometric analysis found that the percentage of hCECs apoptosis induced by MSiNPs-Ag~+was significantly higher than that of MSiNPs at the same concentration.·CCK test found that NSiNPs treatment did not cause cytotoxicity to hCECs,while MSiNPs with the same particle size treated hCECs gradually increased from 100?g/mL concentration,which can produce significant cytotoxicity and there is a significant dose-dependent relationship.When hCECs were treated with MSiNPsAg~+at a concentration of 12.5 ?g/mL,cell viability was significantly reduced.It suggests that MSiNPs have potential corneal cytotoxicity,and after Ag~+adsorption,the corneal cytotoxicity was significantly increased.·The mean fluorescent intensities ofhCECs treated with MSiNPs and MSiNPs-Ag~+were both dramatically higher than that in the control group.Compared with MSiNPs treatment,the increase in ROS levels in the MSiNPs-Ag~+group was more significantly.DNA damage was detected using ?-H2 AX,increased red fluorescence signals were detected in MSiNPs treated cells when compared to the control group while the most significantly enhanced intensity of red fluorescence was observed in MSiNPs-Ag~+exposed group,suggesting that the most severe DNA damages were caused by MSiNPs-Ag~+exposure.·The intracellular mitochondrial damage induced by MSiNPs and MSiNPs-Ag~+treatments were observed by TEM.The significant mitochondrial damage of hCECs was caused by MSiNPs-Ag,and nuclear membrane was severely dissolved and chromatin agglutination.These results suggested that the cytotoxicity of hCECs induced by MSiNPs and MSiNPs-Ag may increase apoptosis through mitochondrial damage,excessive ROS generation,and DNA damage,where MSiNPs-Ag~+could lead to more significant toxic effects than MSiNPs itself.·Compared with the control group,the RNA-seq results indicated that there were 1503 differentially expressed genes(DEGs)inthe MSiNPs group(854 up-regulated and649 down-regulated),and 5157 DEGs in the MSiNPs-Ag~+group(2609 up-regulated and 2548 down-regulated)respectively.·The results of the Kyoto Gene and Genome Encyclopedia(KEGG)pathway and protein-protein interaction analysis indicated that among theDEGs of hCECs treated by MSiNPs,the PI3K-Akt signaling pathway and MAPK signaling pathway are particularly important in induced cytotoxicity.In addition,in the whole genome of hCECs treated with MSiNPs-Ag~+,not only related apoptosis and oxidative stress pathways,but the transcription and translation-associated pathways,mRNA survillance signaling pathway,were identified to be considerably altered.Part ?:Ocularsurface damage and dry eye symptoms when exposed to MSiNPs and MSiNPs-Ag~+and the therapeutic effects of FBS·MSiNPs have excellentadsorption capacity for RhB,NSiNPs have weak adsorption capacity for RhB.This feature confirms that MSiNPs with larger surface area possess better absorbing ability than non-porous NPs.Previously,it is reported that the surface modification of NPs by protein corona can decrease their toxicity.The formation of the protein corona shield could clock the NPs surface and thus inhibit their surface activity.Consistent with RhB result,the intensity of MSiNPs decreased significantly than control and NSiNPs,indicating the effective protein absorption ability compared to control and NSiNPs.Therefore,from these results,we could determine that MSiNPs have the advantage of polydisperse pore size compared to NSiNPs,which could effectively adsorb protein to form protein corona.·This result demonstrated that the repeated short-term exposure of both MSiNPs and MSiNPs-Ag~+ to rats could cause central corneal damage.Among them,MSiNPs-Ag~+induced more serious central corneal injury.Specifically,the corneal damage induced by MSiNPs-Ag~+was greatly reduced by the treatment of FBS.These in vivo results confirmed that the formation of protein corona using FBS treatment could significantly reverse the damage induced by MSiNPs and MSiNPs-Ag~+exposure.·Apoptotic cells in the corne a were labeled with TUNEL staining.Consistent with our in vitro result,a considerably large number of apoptotic cells in the cornea of rats were induced by MSiNPs,while the number of TUNEL-positive cells in the cornea exposed to MSiNPs-Ag~+was even greater than that of MSiNPs(up to 1000 cells).This result provides further evidence that the combination of Ag~+ with MSiNPs could cause a more serious degree of apoptosis than MSiNPs itself.Nevertheless,the FBS treatment obviously reduced the apoptosis caused by NPs treatments(within 100cells),as evidenced by little fluorescence signals of TUNEL-positive cells in the cornea of rat eyes.These cornea damages couldbe reversed by the application of FBS after MSiNPs and MSiNPs-Ag~+ exposure.·Dry eye was tested by TBUT and the Schirmer's test.The MSiNPs-Ag~+ group even showed a greater potential to induce dry eye as TBUTs values in some repeat groups are less than 5secs.Also in the Schirmer's test,Especially at day 7,the Schirmer's test score of MSiNPs-Ag~+ was even less than 5mm,indicating its great possibility to cause dry eye.These results manifest that MSiNPs and MSiNPs-Ag~+ eye drops exposure to rat cornea can induce ocular surface changes similar to those of dry eye in human.After FBS treatment,both TBUTs value(>10 seconds)and Schirmer's test score(>10 mm)are back to normal range in MSiNPs and MSiNPs-Ag~+ exposed groups,further implies the ability of FBS to cure symptoms similar to human dry eye after MSiNPs and MSiNPs-Ag~+ eye drops exposure to the ocular surface.Conclusion:·In this study,we evaluated the synergistic toxicity of MSiNPs with large surface area and Ag~+ on hCECs and rat ocular surface.Most importantly,we also proposed aprotein corona-based therapy to treat MSiNPs induced corneal disease(like Dry eye).·In vitro,the combination of Ag~+ with MSiNPs,even when Ag~+ is applied at a safe dose,resulted in more significant toxicity than MSiNPs itself.RNA-Seq analysis found that MSiNPs may induce cell apoptosis through oxidative stress-drived toxic effects on hCECs.Additionally,MSiNPs-Ag~+ could also have a more serious cytotoxic effect on hCECs by affecting the m RNA monitoring signal pathway.·In vivo,severer corneal damage and symptoms similar to dry eye were caused in ratmodel of short-term repeated ocular surface exposure to MSiNPs-Ag~+ than to MSiNPs.FBS could significantly reverse corneal damage,most likely by forming aprotein corona around the NPs.