Biosafety And Biocompatibility Assessment Of Prussian Blue Nanoparticles In Vitro And In Vivo

As a classic metal-organic framework material,Prussian blue nanoparticles(PBNPs)have good biocompatibility,photothermal and variety of nanozyme activities,which can be widely applied in the medicine.Meanwhile,controllable shape,size and convenient surface modification provide more possibilities for drug delivery and disease diagnosis.However,the morphology,size,and charge of nanomaterials will affect biocompatibility and biosafety in vivo;Moreover,different parameters of nanomaterials have a significant impact on the photothermal and nanozyme activity.Thus,it is necessary to systematically evaluate the biocompatibility and biosafety to of PBNPs with different parameters.In this paper,the stability,photothermal capacity,and the activity of peroxidase-like nanozymes of PBNPs were evaluated in vitro based on different morphologies,charges,and sizes.The hemolysis rate,toxicity,biodistribution,short/long-term toxicity were evaluated in vivo.Although PBNPs showed good biocompatibility and safety in vivo,the liver and kidney toxicity,metabolic rate,photothermal and nanozyme activity can be affected by charge and morphology.The comprehensive exploration of biosafety and biocompatibility provides strong evidence for the application of PBNP as nanodrug carrier.The main results are as follows:The particle size of PBNPs is controlled by changing the concentration of citric acid and reaction time,the morphology is controlled by the hydrochloric acid,and the surface charge is controlled by surface modification.Different shapes,sizes,and surface charges of PBNPs were successfully synthesized,namely NP1(average diameter 30 nm),NP2(average diameter 100nm),NP3(average diameter 200 nm),NC(square nanoparticles),NS(spherical nanoparticles),NP~+(positively charged)and NP~-(negatively charged).By investigating the stability,photothermal,and peroxidase activity,we found that NP3 and NP~+have poor stability in the DMEM and PBS solutions.DLS results showed that NP3 and NP~+particles agglomerated after standing for 5 days,which reduced the stability.In photothermal analysis,NP3 showed the strongest photothermal effect after with laser irradiation.The results showed that particle size increase of NP3 can affect the cross-sectional area of light absorption,thereby enhance the photothermal conversion efficiency.The charge showed great influence on the nanozyme activity as the positive charge on the surface of NP~+can exclude the 3,3',5,5'-tetramethylbenzidine(TMB)of the polyamine group to attract more NP~+into the hydrogen peroxide decomposition.In summary,PBNPs test in vitro proved that the stability,the photothermal effect,and nanozyme activity are related to size and surface charge.2.Biocompatibility and biosafety test of PBNPs assay(1)The average hemolysis rate of all PBNPs at 200?g/ml is 2.35%lower than that of the ISO standard 5%,and less than the hemolysis rate of GO(23%),which showed a better compatibility.(2)MTT assay and Calcein-AM/PI staining results showed that the viability of Hep G2cells treated with NP~+and NP3 decreased to 81%and 58%.(3)In imaging and biodistribution assay in vivo,indicated that the signal of PBNPs quickly accumulated in the liver and kidney at 12 hours and then gradually disappeared 48hours later maybe due to the metabolism through the form of urine.Quantitative analysis showed that the metabolic rate of larger than 50 nm PBNPs(NC,NS,NP2,NP3)was significantly lower than that of smaller than 50 nm PBNPs(NP+,NP-,NP1).NP1 with the smallest size was more likely to be absorbed by the organs of the mononuclear phagocyte system such as liver and kidney.Besides,the fluorescence intensity of NP~+decreased faster than that of NP~-in the same period.This results demonstrated that the interaction between the positive charge and blood proteins can accelerate the clearance of nanomaterials by the liver and kidney.(4)Blood biochemical indicators are used to evaluate the liver and kidney toxicity of PBNPs.Among the liver function markers,alanine aminotransferase(ALT),aspartate amino-transferase(AST),and alkaline phosphatase(ALP)increased significantly at 6 and 12 hours after administration of different doses PBNPs and gradually returned to normal levels.Kidney function indicators creatinine(CREA)and urea(BUN)also returned to normal level followed with the beginning increase.These results indicate that PBNPs can cause transient and slight damage to the liver and kidney.In order to simulate the clinical multi-dose administration method,the effect of PBNPs long-term(2 weeks)on the mice was observed using H&E staining methods.The results showed no accumulation of PBNPs in the important organs.Meanwhile,the liver and kidney function indexes returned to normal,the result of which is similar to that of short-term treatment.In summary,the assay of PBNP in vivo proved its strong blood compatibility and high metabolic efficiency.Size and charge are important factors to affect the rate of metabolism of liver and kidney.