Preparation And Biological Properties Of Lys@C₆₀ Hybrid

C₆₀ has been extensively used in physic,chemistry and material fields due to its unique physical and chemical properties.However,due to its superior hydrophobicity,it tends to aggregate in aqueous solution,hindering the application of C₆₀ in biological fields.Recently,many approaches,such as ultrasonic,functioning with hydrophilic groups,encapsulating with carriers and dispersing with amphiphilic polymers,have been developed to improve the solubility of C₆₀ in aqueous solution.Although these methods solve the water solubility problem of C₆₀ to a certain extent,the monodispersity of C₆₀ has not been achieved yet.At the same time,these methods may bring in new biological toxicity problems.So,it is urged to seek for a new method to address this issue.Based on the excellent biocompatibility,the amphiphilic property,specifically interaction with C₆₀,proteins may be perfect candidates to disperse C₆₀ for biological applications.In this thesis,we propose employing lysozyme to disperse C₆₀by facile ultrasonic and centrifuge treatment,the resulted lysozyme-C₆₀ hybrid(hereinafter referred as Lys@C₆₀)combines the properties of lysozyme and C₆₀,even gains new performance.Then,we explored the possible biological applications of Lys@C₆₀,mainly including the following five aspects:（1）Preparation and characterization of Lys@C₆₀:The monodispersity of C₆₀ was achieved in aqueous solution by simple ultrasonic and centrifugation treatment with lysozyme.The interaction studies showed that it is energy favorable and spontaneously to form Lys@C₆₀ ground state complex in aqueous solution,which is driven by weakπ-πinteraction and hydrogen bonding.Furthermore,thermodynamic studies have shown that the binding and dissociation of C₆₀ with lysozyme could be fine-tuned by temperature.Therefore,lysozyme can be used not only as a“cosolvent”to disperse C₆₀,but also as a carrier to deliver and transport C₆₀ for biological applications.（2）Lys@C₆₀ as an antioxidant for ROS scavenge:Due to the excellent reactive oxygen species（ROS）scavenging ability,C₆₀ is known as a"free radical sponge".We studied the antioxidant properties of Lys@C₆₀ as an antioxidant.The results showed that Lys@C₆₀ has excellent biocompatibility,and could effectively scavenge superoxide anion radicals and hydroxyl radicals,then protect cells against H2O2-induced oxidative stress damage.The studies on the cytoprotection mechanisms elucidated that,the uptake of Lys@C₆₀ into the cell will result in the decrease of the intracellular ROS level,the activation of the cellular antioxidant defense system,and the inhibition of the mitochondrial apoptosis pathway.（3）Lys@C₆₀ as a photosensitizer for antimicrobial photodynamic therapy:C₆₀ not only has the ROS scavenging ability,but also ROS production ability under visible light,so it can be used as a photosensitizer for photodynamic therapy.Together with excellent antibacterial properties of lysozyme itself,we explored the antibacterial photodynamic properties of Lys@C₆₀.The results showed that Lys@C₆₀ combines the antibacterial effect of lysozyme itself with the antibacterial photodynamic effect of C₆₀.The positive Lys@C₆₀ is beneficial to its combination with bacteria,and exerts photodynamic antibacterial properties.This dual antibacterial mechanism could bypass the potential risk of bacterial resistance of Lys@C₆₀.（4）The aggregation-induced emission characteristics of Lys@C₆₀:There is no emission in dilute solutions of Lys@C₆₀ under visible light excitation.However,the enhanced fluorescence is observed in Lys@C₆₀ crystals,which terms as aggregation-induced emission（AIE）.Therefore,the AIE of Lys@C₆₀ was exploited.The results indicated that Lys@C₆₀ does have aggregation luminescence properties revealed by fluorescence microscopy visual observation,photoluminescence spectra of different concentrations of Lys@C₆₀ solution and photoluminescence spectra of Lys@C₆₀aggregation induced by different Na Cl concentrations.The underlying mechanism uncovered by lysozyme crystals shows that the intermolecular and intramolecular interactions of Lys@C₆₀ are enhanced,and the formation of a strong hydrogen bond network greatly stabilize the Lys@C₆₀ molecules,which suppresses the intramolecular vibration and enhances the fluoresence.Based on the aggregation luminescence effect,Lys@C₆₀ can be used as a"fluorescent dye"for cell imaging.（5）The conductivity and ferroelectricity of Lys@C₆₀ crystals:we address the issues of electrode preparation for Lys@C₆₀ crystal by in-situ growth of Lys@C₆₀crystal on conductive substrate,and improve the stability of Lys@C₆₀ crystals for performance testing by cross-linking with glutaraldehyde.The conductivity and ferroelectricity of Lys@C₆₀ detected by AFM showed that the doping of C₆₀ can effectively improve the conductivity and ferroelectric properties of lysozyme crystals.The increasing conductivity of Lys@C₆₀ crystal results from the ordered arrangement of C₆₀ in the lysozyme crystal,which corresponds to p-doping and forms a certain hole and electron pair.When electric field applied,the carrier moves and current forms.The lower symmetry of lysozyme crystal after C₆₀ dopping results in the the ferroelectric properties of Lys@C₆₀ crystal.In summary,this thesis provides a general strategy for the use of protein as“disperser”to improve the solubility of poorly soluble nano-functional materials for biology applications.The strategy makes full use of the excellent biocompatibility,self-assembly characteristics of the proteins,combined with the unique properties of the nano-functional material,the new hybrids gain enhanced performance,even new novel performance.This work not only serves as a platform for"super-biomaterials"synthesis,but also opens up new avenues for the green synthesis of functional materials based on the biocompatible,renewable properties of proteins,which shows great potential applications in biology field.