ACS Appl Mater Interfaces. 2019 Jun 12;11(23):20628-20641. doi: 10.1021/acsami.9b03040. Epub 2019 May 28.
Microcapsules extracted from lycopodium ( Lycopodium clavatum) spores have been increasingly used as an oral therapeutic carrier. A series of sequential treatments involving acetone, KOH, and HPO are used to extract a protein-free hollow microcapsule. This study focuses on two critical aspects of lycopodium spores: the fate of native proteins and the wettability of the spores after a chemical treatment. Protein-free spores are desired to prevent an allergic reaction, whereas the wettability is critical for the formulation development. Although the chemically treated lycopodium spores are generally regarded as protein free, the studies that have reported this have not gone into significant depths to understand the nature of residual nitrogen observed even in spores thought to be protein free. Wettability of spores has not received any significant attention. Accordingly, in this study, we performed a comprehensive analysis of natural spores and spores after each chemical treatment step. We show that natural lycopodium spores are hydrophobic and contain low-molecular-weight proteins (∼10 kD). Acetone treatment partially solubilizes unsaturated phospholipids from the spores. Nevertheless, the acetone-treated spores retain native proteins and are still hydrophobic. KOH treatment, however, removes a significant amount of proteins and partially hydrolyzes esters to carboxylic acid salts and results in a hydrophilic spore with a good wettability. Finally, we show that the HPO treatment removes residual proteins, hydrolyzes remaining esters to carboxylic acids, and dissolves carbohydrates. HPO treatment temperature controls carbohydrate dissolution, which in turn affects the hydroxyl functional groups and hydrophilicity (wettability) of the treated spores. Spores treated at 60 °C as opposed to 160 °C are amphiphilic in nature due to the abundance of hydroxyl functional groups on the surface. In conclusion, this study confirms the removal of native proteins from treated spores and sheds light on the chemical changes that the spores undergo after chemical treatment and correlates these changes to their wettability.
从石松孢子中提取的微胶囊越来越多地被用作口服治疗载体。一系列涉及丙酮、KOH 和 HPO 的连续处理用于提取无蛋白的中空微胶囊。本研究主要关注石松孢子的两个关键方面：天然蛋白质的命运和化学处理后孢子的润湿性。无蛋白孢子是为了防止过敏反应，而润湿性对于制剂开发至关重要。尽管经过化学处理的石松孢子通常被认为是无蛋白的，但已经有研究报告称，即使是那些被认为是无蛋白的孢子中，也观察到了残留氮的存在，但这些研究并没有深入探讨这种情况的本质。孢子的润湿性没有受到任何关注。因此，在本研究中，我们对天然孢子和每个化学处理步骤后的孢子进行了全面分析。我们表明，天然石松孢子是疏水性的，并且含有低分子量蛋白质（约 10 kD）。丙酮处理部分溶解孢子中的不饱和磷脂。然而，丙酮处理的孢子仍保留天然蛋白质且仍疏水性。然而，KOH 处理去除了大量蛋白质，并部分水解酯生成羧酸盐，导致具有良好润湿性的亲水性孢子。最后，我们表明 HPO 处理去除残留蛋白质，水解剩余酯生成羧酸，并溶解碳水化合物。HPO 处理温度控制碳水化合物的溶解，这反过来又影响处理后的孢子的羟基官能团和亲水性（润湿性）。在 60°C 而不是 160°C 下处理的孢子由于表面存在大量羟基官能团而具有两性。总之，本研究证实了处理后的孢子中天然蛋白质的去除，并阐明了孢子在化学处理后经历的化学变化，并将这些变化与其润湿性相关联。
