Study On Protein-polymer Conjugate Particles For Stabilization Of Water-in-water Emulsions And Preparation Of Calcium Carbonate Microspheres

Water-in-water emulsions are formed when two aqueous solutions consisting of incompatible polymers are homogenized,as long as the composition is above the threshold for phase separation.Water-in-water emulsions have some advantages of convenient use,environmental protection and high biocompatibility,which have widely applications in artificial bioreactors,functional materials fabrication and encapsulating and delivering of drugs.However,significant stabilization of w/w emulsions is still a big challenge,due to the ultralow tension of water-water interfaces and the associated large interfacial thickness.As demonstrated in a model system,the bioactive protein particles anchor at the w/w interface,where they can provide a steric barrier against droplet coalescence,not only can stabilize the water-in-water emulsions,but also as a bioactive substance to participate in the reaction.Based on the aggregation mechanism of protein-polymer,we report the preparation of bioactive protein-polymer particles to efficiently stabilize water-in-water emulsions and its application in the enzymatic reaction enhancement and the functional materials fabrication.For bioactive protein particles fabrication:in this part,the growth mechanism of mPEG-protein conjugate particles is proposed.The bioactive protein particles are prepared by methoxy polyethylene glycol acetaldehyde(mPEG-ALD)modify protein at a certain pH and high protein concentration,which efficiently increase the hydrophobicity of protein,and the particles can be obtained by drying at 37 ~oC.During the growth process,there is no addition of any irritant reagents and all reactions are under mild conditions.Using bovine serum albumin(BSA)as the standard protein,we investigate the effect of pH,component concentration and reaction time on the protein particles fabrication.The research indicates that the morphology and structure of mPEG-protein conjugate particles can be controlled by reaction condition such as the pH and drying method.The component concentration and reaction time can affect the size of protein particles.The protein particles size increases as the component concentration is rised or reaction time is prolonged.The isoelectric point(pI)of mPEG-BSA conjugate particles decrease from 4.7 to 4.2 after PEGylation.In addition,as evidenced by enzyme activity assay of urease,the protein particles in our experiment retain at least 80%bioactivity.This research has promising potential application in food industry,cosmetics and biomedicine field.For the stability of water-in-water emulsions:based on the stabilization mechanism of Pickering emulsions,the authors prepare the protein particles with suitable size and wettability by varying the preparation conditions,which can be used to stabilize the water-in-water emulsions and enhance the enzymatic reaction.It has been proved that the mPEG-BSA conjugate particles we use to stabilize water-in-water emulsions are spherical particles with the size of 300 nm and the wettability of 91.6~o.Increasing the particle concentration leads to the formation of stable water-in-water emulsions with decreasing diameters.The two emulsion types(dextran-in-PEG and PEG-in-dextran)can be reversibly switched by varying the volume fraction of the two phases.The pH is an interesting variable affecting the stability of water-in-water emulsions and the emulsions exhibit good stability at pH 4,5 and 9.In addition,the catalytic activity can be enhanced by making the mPEG-urease conjugate particles anchor at the w/w interface.It will have wide applications in life sciences,such as biomedicine and biocatalysis.For functional materials fabrication:using the water-in-water emulsions stabilized by mPEG-urease conjugate particles as a template,the authors prepare uniform calcium carbonate microspheres with controllable morphology via biological mineralization,which can be used as potential bioactive substance delivery carriers.We use the two types of emulsions(dextran-in-PEG and PEG-in-dextran)as templates to prepare uniform calcium carbonate microspheres,which can be used in packaging application for various biological macromolecules.The stability of emulsions can affect the morphology of calcium carbonate microspheres.When ATPS and mPEG-urease conjugate particles are emulsified and then immediately participate in biological mineralization,the peanut-like calcium carbonate microspheres can be obtained.While the emulsions are left standing for a period to obtain stable emulsions and then participate in biological mineralization,the products are spherical calcium carbonate microspheres.It can be found that the calcium carbonate microspheres show a hierarchical structure which consist of flat prismatic calcium carbonate crystals and nano-calcium carbonate particles by scanning electron microscope(SEM).As evidenced by FITC-BSA encapsulation experiment,the calcium carbonate microspheres possess high entrapment efficiency.This study not only provides a good carrier for bioactive substance,but also offers valuable methods and ideas for functional materials fabrication.