Tuning Cellular Oxidative Stress By Diversifying Gold Nanoparticles’ Surface Properties

Due to the outstanding optical,electrical,machanical,and biological properties,nanomaterials are widely used in construction,transportation,energy,chemical engineering,aerospace,personal care products,and medicine.Nanomaterials could enter into human body through respiratory tract,skin,digestive tract and intravenous injection,and distribute into organs and tissues through blood circulation.Nanomaterials may induce various biological effects after interacting with human body,including positive effects,such as disease treatment by nanomedicine,and negative effects,such as nanotoxicity.Therefore,elucidation of the relationship between nanomaterials’ properties and biological effects is crucial for reducing nanotoxicity and extending its application in biomedical field.Reactive oxygen species(ROS)are the deoxidized products of oxygen,including O2-,H2O2,and OH’.ROS could immediately react with biomolecules in cell once formed,resulting in cell damage.In mitochondria,the electrons leaked from respiratory chain could interact with oxygen,leading to the production of ROS.NADPH oxidase is comprised of multiple subunits.In quiescent condition,some subunits are located in cytoplasma,others are located in cell membrane.Under the upstream stimulation,subunits in cytoplasma will translocate into cell membrane,leading to the assembly of all the subunits and activation of NADPH oxidase.NADPH oxidase could catalyze the oxidizing reaction of NADPH,and oxygen will be reduced to ROS.The elevated ROS level leads to oxidative stress,including the overexpression of antioxidant enzyme,inflammation,and cell death.Oxidative stress is correlated to various diseases,including cancer,neurodegenerative disease,and diabetes.Reasonable application of modulators of oxidative stress provides a new strategy for the treatment of these diseases.Therefore,nanomaterials which could modulate oxidative stress have a promising future in the treatment of these diseases.Besides,oxidative stress is the key mechanism of nanotoxicity.Therefore,elucidation of the relationship between nanoparticles’properties and oxidative stress is crucial for disease treatment and reducing nanotoxicity.It was reported that nanomaterials’ composition,size,and shape could tune cellular oxidative stress.Besides,the surface decoration on nanomaterials could also tune the oxidative stress level.Considering that the surface properties of nanomaterials are diverse,there is a lack of systematical investigation between oxidative stress and nanoparticles’ surface properties.And key surface properties which could tune oxidative stress remain unclear.In order to systematically elucidate the impact of surface properties on oxidative stress and identify key surface properties,we designed and synthesized 7 gold nanoparticle(GNP)libraries with each showed a gradient change of a single surface property,including hydrophobicity(HY),positive/negative charge density(PO/NE),hydrogen bond acceptor/donor density(HA/HD),π-bond density(PI),and molecular geometry(MG).The gradient changes of surface properties in PO/NE/HY/HA/HD/PI libraries were achieved through varying the ratio of two kinds of ligands decorated on GNPs in each library.As to MG library,we chose 6 geometrically diverse ligands.The molecular geometry parameters were calculated by computational chemistry method.Each GNP in MG library was decorated with one kind of ligand.The value of molecular geometry parameters of ligands increased with GNPs number.The diameter of synthesized GNPs was about 6 nm,which was characterized by transmission electron microscope(TEM).In order to characterize the surface properties of GNPs,we used I2 to cleave the ligands from the surface of GNPs,and the amount of ligands on each GNP was determined by high-performance liquid chromatography/mass spectrome(HPLC/MS).Based on the amount of ligands on GNPs,we calculated the change of surface properties for PO/NE/HA/HD/PI/MG libraries.The positive/negative charge density,hydrogen bond acceptor/donor density,π bond density,and molecular geometry parameter increased with GNPs’ number in these libraries.As to HY library,we used modified shaking flask method to determine the experimental LogP value.The hydrophobicity increased with GNPs’ number in HY library.The characterization data demonstrated that we have successfully synthesized 7 GNP libraries each exhibiting a gradient change in a specific surface property.We evaluated oxidative stress induced by GNPs with different surface properties in multiple human cells.The correlation analysis between intracellular heme oxygenase 1(HO-1)/H2O2 level and GNPs’ surface properties demonstrated that positive charge density and hydrophobicity were positively correlated with oxidative stress level,while other properties showed no correlation.In multiple human cells,the cellular oxidative stress level increased with hydrophobicity and positive charge density.GNPs could adsorb protein in blood,which may affect its biological activity,such as immune response and cytotoxicity.We compared the intracellular H2O2 levels induced by GNPs in PO and HY libraries in cell culture medium with different serum protein concentrations.The results demonstrated that oxidative stress level induced by GNPs was identical when serum protein concentrations were 10%and 50%.Therefore,oxidative stress induced by GNPs was independent of serum protein concentrations.Oxidative stress induced by nanomaterials is usually accompanied with cellular uptake.However,for nanomaterials with specific surface properties,the dependence of oxidative stress on cellular uptake remains unclear.Based on TEM image and inductively coupled plasma mass spectrometry(ICP-MS)characterization,we found that oxidative stress level induced by GNPs in PO and HY libraries was positively correlated with cellular uptake.To establish a dependence of oxidative stress on cellular uptake,we used cytochalasin D(Cyto D)to inhibit the cellular uptake of positively charged and hydrophobic GNPs.The reduction of cellular uptake led to the decrease of intracellular H2O2 level,demonstrating that endocytosis was required for oxidative stress generation for positively charged and hydrophobic GNPs.Mitochondria and NADPH oxidase are two main sources of oxidative stress.First,we employed a chemical—biological method to distinguish the oxidative stress sources of hydrophobic and positively charged GNPs by using mitochondria and NADPH oxidase inhibitors.Western blot and fluorescence experiments were further used to elucidate the mechanisms.NADPH oxidase inhibitors could totally inhibit the oxidative stress induced by hydrophobic GNPs,indicating that hydrophobic GNPs may induce oxidative stress through NADPH oxidase pathway.The translocation of subunits in cytoplasma represents the activation of NADPH oxidase.The results of western blot experiments indicated that after treatment of hydrophobic GNP,the cytoplasma subunit p47phox translocated to cell membrane.For 3 different cell lines,the p47phox level on cell membrane was positively correlated with intracellular H2O2 level after hydrophobic GNP treatment.Therefore,hydrophobic GNPs induced oxidative stress through NADPH oxidase activation.As to positively charged GNPs,the mitochondria respiratory chain complex 1 inhibitor could totally inhibit the oxidative stress induced by positively charged GNPs.Therefore,we speculated that positively charged GNPs may induce oxidative stress through mitochondria pathway.The results of western blot and fluorescence experiments demonstrated that positively charged GNPs induced cell membrane depolarization,which finally led to oxidative stress.Both small molecules and biomolecules could enhance the effects of chemotherapeutics through eliciting oxidative stress.Based on the results mentioned above,we speculated that oxidative stress induced by hydrophobic and positively charged GNPs could also enhance the effects of chemotherapeutics.The results demonstrated that hydrophobic and positively charged GNPs could enhance the cytotoxicity of paclitaxel in HeLa cells.The ROS scavenger N-acetyl-L-cysteine(NAC)could effectively alleviate the oxidative stress level induced by GNPs,resulting in the decrease of cytotoxicity induced by the mixture of paclitaxel and GNPs.Therefore,hydrophobic and positively charged GNPs could enhance the effects of chemotherapeutics through eliciting oxidative stress.Based on the systematical investigation about oxidative stress induced by GNPs with diverse surface properties,we found that hydrophobicity and positive charge density are key properties which could tune oxidative stress,The elucidation of oxidative stress related mechanisms induced by nanomaterials with different surface properties could precisely guide the application of nanomaterials in disease treatment and regulation of nanotoxicity.We also found that hydrophobic and positively charged GNPs as a potential chemosensitizer could enhance the effects of chemotherapeutics in vitro,which afforded new strategies for the application of nanomaterials in cancer therapy.