Pickering Emulsion Templated Proteinaceous Microsphere With Bio-stimuli Responsiveness

In recent years,microspheres that respond to biological signals in the tumor microenvironment have received increasing attention.Bioresponsive microspheres can take advantage of differences at the tumor site to achieve precise drug release.Many attempts have been made to explore bioresponsive microspheres,most of which rely on synthetic polymers,leading to a series of health issues that seriously limit their biomedical applications.In contrast,biopolymers have attracted attention for their biocompatibility,biodegradability,low antigenicity,and high biological activity.Therefore,this paper focuses on the preparation of zein-based microspheres from natural sources and their bioresponsive release.The main research contents are as follows:（1）Controllable fabrication of SiO₂/zein hybrid microspheresSilica nanoparticles of various sizes were synthesized by the St（?）ber method and modified by reacting with dichlorodimethylsilane.The Pickering emulsions were stabilized by hydrophobic silica nanoparticles,while zein protein was dissolved in the middle phase.Subsequent ethanol removal from the emulsion template precipiated the protein skeleton.Firstly,we stained the aqueous ethanol phase and the oil phase to demonstrate that the type of emulsion was oil-in-（ethanol/water）-in-oil.Then,the formation mechanism of Pickering multiple emulsions was explored,and the research results showed that it was mainly formed by the phase inversion of emulsions stabilized by zein.Finally,the morphology of the emulsion template and microspheres was controlled by various factors such as zein concentrations,oil/（ethanol-water）volume ratios,emulsifier concentrations,particle sizes,and different types of particles.（2）Study on release of proteinaceous microsphere with bio-stimuli responsivenessBased on the fabrication of SiO₂/zein hybrid microspheres mentioned above,fluorescein isothiocyanate（FITC）-labeled dextran was chosen as the model active for encapsulation into microspheres.The results showed that it was uniformly dispersed in the protein matrix without affecting the structure of the microspheres.On this basis,it was noted that the structure was intact under a relatively wide pH range（3-11）,and thus,it has a high resistance.Finally,we investigated the bio-stimuli-responsive behavior of microspheres.The results showed that the proteinaceous microspheres have high encapsulation efficiency of model actives and have shown excellent bio-stimuli-responsiveness to glutathione（GSH）and protease.（3）GSH-response-induced death of cancer cells based on TA/zein hybrid microspheresTo address the issue of poor dispersion of the aforementioned hybrid microspheres in water medium,hydrophobic SiO₂ particles were replaced with hydrophobic Ca CO₃ particles.The Ca CO₃ on the surface of the microspheres was then etched using TA to facilitate their dispersion in culture medium.Suitable microsphere sizes for subsequent cell uptake were obtained by controlling emulsification method,oil phase type,particle concentration,and oil/（ethanol-water）volume ratio.Subsequently,the morphology and composition of TA/zein hybrid microspheres were characterized,and in vitro GSH-responsive release was studied.Finally,He La cell uptake of the microspheres at different time was investigated,and the fluorescence dye was replaced with doxorubicin（DOX）.Cell viability was tested through MTT assay,and the results showed a decreasing trend in cell viability with increasing time and concentration of the microspheres.