Preparation Of Functionalized Zein-based Paclitaxel Delivery System And Its Performance Study

Zein is a natural,biocompatible and biodegradable renewable material.Zein has beeninvestigated as various carriers,such as micro and nano particles,films and fibers,because of its unique amphiphilic and self-assembly characteristics.It is widely used in pharmaceutical fields including drug targeting,vaccines,tissue engineering and gene delivery.However,zein based drug delivery system（DDS）lacks the ability to target and immune escape,which would difficult to accumulate at the lesion and easy to be cleared by the immune system.Further more,zein is a mixed protein,and the mechanism of its modification is not clear.Therefore,isolation and purification of zein are necessary for its future development.In this dissertation,paclitaxel（PTX）,a broad-spectrum anticancer drug,was chosen as the model drug.Zein was modified and purified to prepare safe and functional zein-based DDS,the modern advanced characterization technologies were used to study the drug loading,the co-assembly principle of drug carriers and the performance of DDS.The research contents of this dissertation include:（1）Enhancing hemocompatibility and the performance of Au@SiO₂ nanoparticles by coating with functionalized zein.It was found that the Au@SiO₂ sample caused serious hemolysis when its concentrations were>200μg/m L,the hemolysis rate reached 107%and spiculated cells were found.After coating with the functionalized zein,the samples showed a low hemolysis rate from 1.4 to 4.0%in the concentration ranges from 25 to 800μg/m L,and the morphology of RBCs showed no significant change.The strong interaction between Au NPs and cysteine residues resulted in the conformation change of functional zein.The hemolysis behaviors did not occur because the active site of Au NPs was occupied by functional zein.After coating with cRGD-Zein,the drug release was reduced to 13%at 2 h and the burst release never occurred from 2 to 48 h.The results indicated that cRGD-Zein could effectively avoid premature drug leakage and realize sustained release.The cell cytotoxicity and uptake assays showed that the DDS exhibited low tumor cell viability（35%）and enhanced uptake performance（99.3%）.（2）Purification of zein,analysis of the differences in self-assembly behavior,molecular structure and drug delivery performance betweenαandβ-Zein.Theαandβ-zein were separated according to different solubility.The different self-assembly behavior and mechanical properties were investigated by AFM method at nano scale before and after drug loading.The results showed thatα-zein nanoparticles self-assembled in the ethanol possessed a higher adhesion force（99.45?6.90 n N）,which explained the reason for the uncontrollable self-assembly and explosive nucleation ofα-zein.After loading with PTX,Young’s modulus ofα-zein significantly reduced from 525.57?53.82 to 95.35?20.98 MPa,which implied that the PTX-α-zein nanoparticle had a loose structure.The interaction between drug and carrier was studied by using a fluorescence quenching experiment,and the mathematical model was established.The result showed that the main driving force for interaction between PTX andα-zein was the hydrophobic force,while interaction between PTX andβ-zein was hydrogen bonding and Van der Waals forces.The negative（35）G value indicated that the binging between PTX andα-zein orβ-zein was a spontaneous process although the nature of the interaction was somewhat different.The in vitro cytotoxicity study showed that the different self-assembly behavior resulted in different cell viability.Finally,a possible self-assembly mechanism ofαandβ-zein was proposed.（3）Preparation and performance study of cell membrane coatedα-zein biomimetic nano-drug loading system.A solvent is key for preparingα-zein nanoparticles,the appropriate solvents were selected to study the effects of different solvents on the solubility and self-assembly behavior ofα-zein,and the particle size uniformity,stability and toxicity were evaluated.Then B16 cancer cell membrane（CCM）modifiedα-Zein based nanocarrier was prepared using a co-extrusion technique.The successful preparation of DDS was proved by AFM,FTIR and zeta potential.The results of SDS-PAGE protein electrophoresis showed that CCM-α-zein could completely retain the proteins ofα-zein and CCM after extrusion fusion.The safety of the prepared DDS was proved by the hemolysis test and non-tumor cells experiment.NMR,XRD and TG-DSC were used to characterize the drug loading behavior,and the appropriate drug proportion was optimized.The results of the cell experiment showed that the migration surface of B16 cells was reduced by 5.79%,and the clone formation rates of A549,Hela and B16 were 57.8%,67.1%and 0%,respectively,which indicated that the inhibition ability for B16 cells was relatively strong.The results of nuclear activity test showed that the nuclei of B16 cells were broken into fragments.Qualitative and quantitative studies showed that the drug carrier coated with B16 CCM had homologous targeting ability.