Modulating The Bioactivity Of Protein-nanoparticle Conjugates

The modulation of protein activity is of great significance for disease therapy,molecular diagnosis,and tissue engineering.The nanoparticle offers a new platform for proteins conjugated on its surface and bioactivity modulation,showing considerable potential in areas such as controlled delivery and release of drugs,biosensing,and biocatalysis.The main focus of this thesis is to fabricate multifunctional protein-nanoparticle conjugates and investigate on the mechanism and effects of activity-modulating capability of the conjugates systematically.The detailed works are introduced as below.Firstly,two mutant types(MTs)of Escherichia coli inorganic pyrophosphatase(PPase)were generated through site-directed mutagenesis with a single Cys residue introduced on the protein surface.The effect of protein orientation on the bioactivity of conjugates was studied by comparing MTs with wild type PPase(WT)conjugated to gold nanoparticles(AuNPs).Meanwhile,the effect of surface density of proteins on the bioactivity of conjugates was investigated by controlling the original feed molar ratio of CPPase/CAuNP.The results demonstrated that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density.Site-directed orientation with the attachment site far from the active center allows virtually complete retention of activity.Furthermore,increasing the surface density of this specifically oriented protein allows increased loading of the protein without affecting the activity to any significant extent;the amount of proteins conjugated on the AuNP also increases,that is the increasing the total bioactivity per AuNP.This strategy is of great importance in the applications for enzyme immobilization,drug delivery,and biocatalysis.However,this work still has some limitations.Once protein-nanoparticle conjugates are prepared,the bioactivity of protein in the conjugates is difficult to further modulate.In addition,proteins located on the outside of the conjugates are mostly exposed to the environment,leading to easy proteolysis by proteases.Therefore,it is essential to establish a new protein-nanoparticle conjugate system for better bioactivity modulation.Secondly,PPase and poly(N-isopropylacrylamide)(PNIPAM)were both fabricated onto AuNPs,forming AuNP-PPase-PNIPAM conjugates.We illustrated activity-modulating capability of conjugates via modulating steric hindrance of PNIPAM to proteins incorporating sensitivity to temperature.The activity-modulating capability of the conjugates with changes in temperature was systematically detected by varying the molecular weight of PNIPAM,the PPase/PNIPAM molar ratio on AuNP,and the orientation of the proteins.The tolerance to trypsin digestion of the native PPase and the conjugates was studied at different temperatures.The results demonstrated that the conjugates can both modulate protein activity drastically and produce a much higher resistance to trypsin digestion.This strategy shows great potential in the areas of targeted delivery,controllable biocatalysis,and molecular/cellular recognition.However,the steric hindrance of polymers to proteins limits the range of activity modulation and the extent of resistance to proteases.And the procedures to prepare the nanoparticle–protein conjugates are too complicated to be reused and recycled.Therefore,it is in urgent need to solve these problems and improve the stragety.Finally,based on the above considerations,we fabricated PPase and poly(methacrylic acid)(PMAA)on AuNPs,forming AuNP-PPase-PMAA conjugates.The bioactivity of the AuNP-PPase-PMAA conjugates was modulated by treating with poly(2-(dimethylamino)ethyl methacrylate)(PDMAEMA)and regulating pH.The activity-modulating capability was systematically studied by varying the molecular weights of the PMAA and PDMAEMA,particle size and the PPase/AuNP feed molar ratio.The tolerance to trypsin digestion of the conjugates was investigated at different pH.In contrast to the simple shielding effect afforded by a single polymer type,the interactions between two pH-responsive polymer types,as shown in this work,give a better shielding effect and much greater resistance to protease attack.The fabrication of the conjugates can realize “on/off”-switchable bioactivity modulation;in addition,by taking advantage of the ability of AuNPs to undergo reversible aggregation/dispersion,the conjugates can be recycled and reused multiple times;and due to the shielding effect of the PMAA,the conjugated enzyme has higher resistance to protease digestion.This strategy has considerable potential in areas such as controlled delivery and release of drugs,biosensing,and biocatalysis.In conclusion,we developed different and efficient strategies to modulate bioactivity of protein-nanoparticle conjugates,from controlling site-directed orientation and surface density of proteins to introducing temperature-responsive polymers and taking advantage of the interactions between two types of pH-responsive polymers.Finally,protein-nanoparticle conjugates could realize “on/off”-switchable bioactivity modulation by incorporating sensitivity to environmental cues,recyclable use and enhanced resistance to protease digestion.The multilevel,multifunctional and accurate bioactivity modulation of protein-nanoparticle conjugates have great potential in the areas of drug delivery,biosensors,biocatalysis and environmental treatment.