Assessment Of Three Nanoparticals On Acute Ocular Toxicity And Biosafety In Mice

Nanomaterials play an important role in the fields of medicine,home appliances,textiles,electronics industry,environmental protection and machinery independent on its unique physical and chemical properties.However,nanomaterials will inevitably be released into the atmosphere and directly contacted with humans and other organisms with their production,application,and emission,which will cause potential ecological biological health risks.This study is mainly focused on the biosafety assessment of nanomaterials,we have compared three nanoparticles and analyzed their characterizations.Graphene oxide(GO),reduced graphene oxide(RGO)and polyethylene glycol(PEG)coated boron nitride(PEG-BNs)were used in this study,between which share the similar structures and have been regarded as tested meterials.We used kunming mice as tested animals.The acute ocular toxicities of those three nanoparticles were investigated on the level of both morphology,pathology and biochemistry via exposing to ocular surface respectively and intravitreal injection.Biodistributions of PEG-BNs were detected using a radioactive isotope tracer method.Toxicity of PEG-BNs and the healing effect of simvastatin were studied by taking advantage of pathological and biochemical methods.i.GO,RGO and PEG-BNs were prepared respectively using chemical REDOX method and chemical modified method.Structural analysis of the samples were performed by TEM,FTIR,XRD,Raman spectroscopy and TGA.The results showed that GO and RGO nanoparticles of which sizes were about 100 nm had been successfully prepared by using chemical REDOX method.Oxygen-containing groups including hydroxy and carbonyl were added onto the surface of GO,but there was no oxygen-containing groups on RGO surface.Because of the existence of these new groups,the layer spacing of GO was wider than that of RGO(layer spacing of GO was about 0.707 nm and that of RGO was about 0.335 nm).And GO also showed a more disordered structure than that of RGO(ID/IG of GO was about 1.21 and that of RGO was about 0.89).In the mean time,PEG-BNs with the size of about 20 nm was successfully prepared via using chemical modified method,and quality ratio of PEG was up to 99.4% with PEG high modificational degree.Besides,PEG-BNs showed the similar structure of BNs and good water solubility.ii.The different concentrations(25,50 and 100 ?g/mL)of solutions/suspension of RGO,GO and PEG-BNs were prepared using 0.01 M PBS buffer,then 10 ?L of RGO,GO and PEG-BNs solution were exposed to the right conjunctival sac of mice of experimental groups respectively,the mice of control group were performed to PBS buffer only;animals of all groups were subjected to the exposure operation for 7 days,1 time/day.Different levels of cornea opacity have been detected to be appeared after GO exposure for 7 days,and such opacity altered in dependent with the concentration of GO,however,no significant difference has been detected in the mice exposed to RGO and PEG-BNs of three concentrations respectively.Furthermore,the corneal opacity could be found to be more obvious in the animals of which eyes had been treated with low concentration of GO(25 ?g/mL)for 10 days.Syndromes including corneal lesions,incrassated corneal stromal layer,increased corneal stromal cell density,obvious intraocular inflammation,cell apoptosis in the cornea and iris neovascularization have all been detected in the mice of which eyes had been exposed to GO.On the contrary,those syndromes mentioned above have not been detected in the mice of both RGO-and PEN-BNs-operated group.In the mean time,expression levels of inflammatory factors such as TNF-?,IL-6,IL-8 and IL-10 have been up-regulated in the mice of GO-operated group,and the expression levels of such forementioned factors were examined to be had no significant difference in RGO and PEN-BNs-operated group.The increased level of MDA in the animals of GO group indicated that peroxidation was occured in eyes,and the increased level of T-AOC represented that the body's natural antioxidant mechanisms in eyes has been induced,while the level of MDA/T-AOC in the mice of RGO and PEN-BNs groups remained unchanged.Further,to investigate whether GO can pierce cornea the enter into the aqueous humor,the eyeballs of the mice were extracted and isolated from cornea,the the eyeballs were digested with strong acid under high temperature,then the GO lamer has been found the in the product under TEM,which indicated that GO could pierce cornea and enter into the aqueous humor.iii.The solutions/suspension of RGO,GO and PEG-BNs were dissolved with 0.01 M PBS buffer respectively into concentrations of 25 ?g/mL,and then the mice eyes were exposed to 2 ?L of the solution/suspension respectively using intravitreous injection,the mice eyes of control group were performed to PBS buffer exposure.At the first day after GO exposure,the rods and cones layer(RCL),the inner plexiform layer(IPL)were significantly thinned,the inner nuclear layer(INL)and outer plexiform layer(OPL)were significantly thickened;at the third day after exposure the thicknesses of RCL,IPL,INL and OPL have been recovered significantly;the RCL,IPL,INL and OPL could be restored to have no significant difference with those of the the mice in control group after 5~7 days.However,the thickness of outer nuclear layer(ONL)and retinas were remained at normal levels during the exposure-recovery progression.While no lesion was presented in the pathological section of the retina during 7 days after RGO and PEG-BNs exposure.Moreover,at the first and third day after GO exposure,the levels of both of MDA and T-AOC were significantly increased;then the levels of MDA/T-AOC were recovered to normal levels after 7 days.The levels of MDA and T-AOC in eyes exposed to RGO and PEG-BNs showed no significant difference during 7 days.Such results indiated that GO could cause transient reversible retinal injury,but RGO and PEG-BNs showed no significant effect on retina.iv.As can be seen from the above,RGO and PEG-BNs show good ocular compatibility,but compared with RGO,PEG-BNs has the advantages of small size and high solubility,etc.,therefore the acute toxicity of PEG-BNs and it's prevention and treatment effects were invesgated fuether in vivo.Different concentrations of PEG-BNs were intravenously injected into mice,then the biodistribution of PEG-BNs has been investigated using radioisotope tracers.It has been found that PEG-BNs were mainly distributed in the liver,spleen and lung;few level of PEG-BNs has been detected in heart and kidney respectively.The highest level of PEG-BNs distribution in the organs has been detected at the first hour after exposure,and the level distribution in heart,lung and spleen decreased with time going;however,the distribution of PEG-BNs in blood,liver and kidney firstly reduced and then increased.After 24 h,the existance of PEG-BNs could still be detected in these organs and the levels of biochemical indicators(AST,ALT,TB,CREA,BUN and Cys-C)in serum have changed,meanwhile,significant pathological changes have been presented in the organs.SST showed a good therapeutic effect to the syndromes caused by PEGBNs nanoparticles,but showed no preventional and synergistic effects.In this study,the acute ocular toxicity of GO,RGO and PEG-BNs nanoparticles were quantitatively analyzed,the results indicated that RGO and PEG-BNs showed good ocular compatibility,which could be used in clinical ophthalmology as the new types of nano-medical materials.However,GO could cause severe eye damage by directly contacting with eyes,which indicated that increasing level of environmental GO nanoparticles may have potential damage to the eyes.Besides,the biodistributions and the toxicity of PEG-BNs nanoparticle have been investigated,so as the prevention and therapeutic effects to the damage,which have provided a new reference and new thoughts to this field.