Wu Changzhu, Böttcher Christoph, Haag Rainer
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany.
Soft Matter. 2015 Feb 7;11(5):972-80. doi: 10.1039/c4sm01746c.
The enormous potential of nanogel scaffolds for protein encapsulation has been widely recognized. However, constructing stable polymeric nanoscale networks in a facile, mild, and controllable fashion still remains a technical challenge. Here, we present a novel nanogel formation strategy using horseradish peroxidase (HRP) catalyzed crosslinking on phenolic derivatized dendritic polyglycerol (dPG) in the presence of H2O2 in an inverse miniemulsion. This "enzymatic nanogelation" approach was efficient to produce stable 200 nm dPG nanogel particles, and was performed under physiological conditions, thus making it particularly beneficial for encapsulating biological proteins. Purification of the nanogels was easy to handle and practical because there was no need for a post-quenching step. Interestingly, the use of dPG resulted in higher HRP laden nanogels than for linear polyethylene glycol (PEG) analogs, which illustrates the benefits of dendritic backbones in nanogels for protein encapsulation. In addition, the mild immobilization contributed to the enhanced thermal stability and reusability of HRP. The nanogel preparation could be easily optimized to achieve the best HRP activity. Furthermore, a second enzyme, Candida antarctica lipase B (CalB), was successfully encapsulated and optimized for activity in dPG nanogels by the same enzymatic methodology, which shows the perspective applications of such techniques for encapsulation of diverse proteins.
纳米凝胶支架在蛋白质封装方面的巨大潜力已得到广泛认可。然而，以简便、温和且可控的方式构建稳定的聚合物纳米级网络仍然是一项技术挑战。在此，我们提出了一种新型的纳米凝胶形成策略，即在反相微乳液中，利用辣根过氧化物酶（HRP）在H2O2存在的情况下催化酚类衍生化树枝状聚甘油（dPG）交联。这种“酶促纳米凝胶化”方法能够高效地制备出稳定的200 nm dPG纳米凝胶颗粒，并且是在生理条件下进行的，因此对于封装生物蛋白质特别有利。纳米凝胶的纯化易于操作且实用，因为无需进行后淬灭步骤。有趣的是，与线性聚乙二醇（PEG）类似物相比，使用dPG可得到负载更多HRP的纳米凝胶，这说明了树枝状主链在用于蛋白质封装的纳米凝胶中的优势。此外，温和的固定化有助于提高HRP的热稳定性和可重复使用性。纳米凝胶的制备可以很容易地进行优化以实现最佳的HRP活性。此外，通过相同的酶促方法，第二种酶——南极假丝酵母脂肪酶B（CalB）成功地被封装在dPG纳米凝胶中并对其活性进行了优化，这展示了此类技术在封装多种蛋白质方面的潜在应用。
