Pickering乳液模板构建生物刺激响应性蛋白质微球

doi:10.3866/PKU.WHXB202301041

摘要/Abstract

摘要：

生物刺激响应性微球可用于封装活性物，并在生理条件刺激下触发释放活性物，在制药，化妆品，食品和农业等领域备受关注。然而大多数微球材料基于合成聚合物，合成过程中涉及的有机溶剂或交联剂残留易产生安全性与生物相容性隐患。因此，本工作以Pickering双重乳液为模板，探索一种简单、可持续地构建生物刺激响应性蛋白质微球的方法。具体地说，本文采用Stöber法合成了粒径约为100 nm的二氧化硅颗粒，并用二氯二甲基硅烷对其进行疏水改性。随后，Pickering双重乳液模板由疏水二氧化硅纳米颗粒稳定，玉米醇溶蛋白溶解在中间相。同时，乙醇的移除过程促使蛋白质析出并构成微球骨架。我们先通过染色法，分别用罗丹明B标记乙醇水相和芘标记油相，通过激光共聚焦(CLSM)证明了多重乳液的结构。并通过扫描电子显微镜(SEM)详细表征了蛋白微球和疏水二氧化硅颗粒的形貌，我们发现Pickering乳液模板制备的微球具有良好的球形度和均一性，且二氧化硅颗粒在微球表面均匀排列，其中疏水二氧化硅颗粒除了充当乳化剂外，还能提高蛋白微球的机械强度和单分散性。紧接着，我们以FITC标记的葡聚糖作为封装模型，通过CLSM证明了FITC-葡聚糖在蛋白微球中均匀分散且对微球结构没有影响。之后我们考察了蛋白微球的pH耐受性，通过光学显微镜发现，蛋白微球在pH 3–11条件下结构保持良好，只有在pH大于11的情况下才会分解释放，证明其具有良好的耐受性。最后，我们探索了蛋白微球的生物刺激响应行为，由于玉米醇溶蛋白含有大量的硫氨基酸，可形成分子内和分子间二硫键，而二硫键可以被谷胱甘肽(GSH)还原，且蛋白质本身具有酶解特性，因此该蛋白微球同时具有GSH和酶响应性，用荧光分光光度计测量了蛋白微球在不同GSH和酶浓度下FITC-葡聚糖的释放量，且由CLSM和SEM进一步验证了微球的分解行为。综上，Pickering乳液模板的良好稳定性和可调控性使蛋白质微球具有结构可控性。此外，结果显示该蛋白质微球对活性物有良好的保护与包封效果，且对蛋白酶和谷胱甘肽表现出非凡的生物刺激响应性。

关键词: 生物刺激响应, 蛋白质微球, Pickering乳液, 蛋白酶, 谷胱甘肽

Abstract:

Bio-stimuli-responsive microspheres, which can encapsulate and release actives in response to physiological triggers, have attracted increasing attention in pharmaceutical, cosmetic, food biotechnology, and agricultural industries. However, most microspheres are based on synthetic polymers and suffer from a lack of biocompatibility due to the residues of harsh organic solvents or crosslinkers used in the synthesis process. Herein, we develop a simple and sustainable method for the construction of proteinaceous microspheres templated from Pickering double emulsions. Specifically, silica nanoparticles with a diameter of 100 nm were synthesized by Stöber method and modified by reacting with dichlorodimethylsilane. The Pickering emulsions are stabilized by hydrophobic silica nanoparticles, while zein protein is dissolved in the middle phase. Subsequent ethanol removal from the emulsion template precipitated the protein skeleton. First, we stained the aqueous ethanol phase with rhodamine B and the oil phase with pyrene to demonstrate the formation of double emulsions by confocal laser scanning microscopy (CLSM). The morphology of microspheres and silica nanoparticles was characterized by scanning electron microscopy (SEM). The obtained microspheres showed high sphericity and uniformity. In addition to acting as particulate stabilizers, the silica nanoparticles could improve the mechanical strength and monodispersity of microspheres. Herein, fluorescein isothiocyanate (FITC)-labeled dextran was chosen as the model active for encapsulation into microspheres. The CLSM images showed that it was uniformly dispersed in the microspheres and had no effect on the structure of the microspheres. Next, we investigated the pH tolerance of the microspheres. Through optical microscope, it was noted that the structure was intact under pH 3–11, and thus, it has a high resistance. Finally, we investigated the bio-stimuli-responsive behavior of microspheres. Zein is rich in sulfur-containing amino acids, which can form intra- and inter-molecular disulfide bonds. Because disulfide bonds can be reduced by glutathione (GSH) and the protein itself has enzymatic hydrolysis characteristics, the proteinaceous microspheres can be triggered release in response to GSH and protease. The release profiles of FITC-dextran from microspheres at different concentrations of GSH and protease were evaluated by fluorescence spectrophotometer. The decomposition behavior of microspheres under certain concentrations of GSH and protease was further verified by CLSM and SEM. To conclude, excellent stability and tunability of emulsion templates render the resulting proteinaceous microspheres with adjustable structures. Meanwhile, the proteinaceous microspheres have high encapsulation efficiency of model actives and have shown excellent bio-stimuli-responsiveness to protease and glutathione.

Key words: Bio-stimuli-responsive, Proteinaceous microsphere, Pickering emulsion, Protease, Glutathione

蒋伟杰, 蒋航, 刘威, 管鑫, 李云兴, 杨成, 魏涛. Pickering乳液模板构建生物刺激响应性蛋白质微球[J]. 物理化学学报, 2023, 39(12), 2301041. doi: 10.3866/PKU.WHXB202301041

Weijie Jiang, Hang Jiang, Wei Liu, Xin Guan, Yunxing Li, Cheng Yang, To Ngai. Pickering Emulsion Templated Proteinaceous Microsphere with Bio-Stimuli Responsiveness[J]. Acta Phys. -Chim. Sin. 2023, 39(12), 2301041. doi: 10.3866/PKU.WHXB202301041

链接本文: https://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB202301041

https://www.whxb.pku.edu.cn/CN/Y2023/V39/I12/2301041

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