Nanoly Bioscience, Inc. , Denver , Colorado 80231 , United States.
Macromolecular Engineering Laboratory, Department of Mechanical and Process Engineering , ETH Zürich , 8092 Zürich , Switzerland.
Biomacromolecules. 2018 Mar 12;19(3):740-747. doi: 10.1021/acs.biomac.7b01507. Epub 2018 Feb 9.
Modern medicine, biological research, and clinical diagnostics depend on the reliable supply and storage of complex biomolecules. However, biomolecules are inherently susceptible to thermal stress and the global distribution of value-added biologics, including vaccines, biotherapeutics, and Research Use Only (RUO) proteins, requires an integrated cold chain from point of manufacture to point of use. To mitigate reliance on the cold chain, formulations have been engineered to protect biologics from thermal stress, including materials-based strategies that impart thermal stability via direct encapsulation of the molecule. While direct encapsulation has demonstrated pronounced stabilization of proteins and complex biological fluids, no solution offers thermal stability while enabling facile and on-demand release from the encapsulating material, a critical feature for broad use. Here we show that direct encapsulation within synthetic, photoresponsive hydrogels protected biologics from thermal stress and afforded user-defined release at the point of use. The poly(ethylene glycol) (PEG)-based hydrogel was formed via a bioorthogonal, click reaction in the presence of biologics without impact on biologic activity. Cleavage of the installed photolabile moiety enabled subsequent dissolution of the network with light and release of the encapsulated biologic. Hydrogel encapsulation improved stability for encapsulated enzymes commonly used in molecular biology (β-galactosidase, alkaline phosphatase, and T4 DNA ligase) following thermal stress. β-galactosidase and alkaline phosphatase were stabilized for 4 weeks at temperatures up to 60 °C, and for 60 min at 85 °C for alkaline phosphatase. T4 DNA ligase, which loses activity rapidly at moderately elevated temperatures, was protected during thermal stress of 40 °C for 24 h and 60 °C for 30 min. These data demonstrate a general method to employ reversible polymer networks as robust excipients for thermal stability of complex biologics during storage and shipment that additionally enable on-demand release of active molecules at the point of use.
现代医学、生物研究和临床诊断依赖于复杂生物分子的可靠供应和储存。然而，生物分子本质上容易受到热应力的影响，而包括疫苗、生物疗法和研究用仅（RUO）蛋白在内的高附加值生物制剂的全球分布需要从制造点到使用点的综合冷链。为了减少对冷链的依赖，已经设计了制剂来保护生物分子免受热应力的影响，包括基于材料的策略，通过直接封装分子来赋予热稳定性。虽然直接封装已经证明了对蛋白质和复杂生物流体的显著稳定作用，但没有一种解决方案既能提供热稳定性，又能方便地按需从封装材料中释放，这是广泛应用的关键特征。在这里，我们展示了在合成的光响应水凝胶内直接封装可以保护生物分子免受热应力的影响，并在使用点提供用户定义的释放。基于聚乙二醇（PEG）的水凝胶是通过在存在生物制剂的情况下进行生物正交的点击反应形成的，而不会影响生物制剂的活性。安装的光不稳定部分的裂解使随后用光溶解网络并释放封装的生物制剂成为可能。水凝胶封装提高了常用于分子生物学的封装酶（β-半乳糖苷酶、碱性磷酸酶和 T4 DNA 连接酶）在热应力后的稳定性。β-半乳糖苷酶和碱性磷酸酶在高达 60°C 的温度下稳定 4 周，在 85°C 下稳定 60 分钟用于碱性磷酸酶。T4 DNA 连接酶在中等升高的温度下迅速失去活性，在 40°C 的热应力下保护 24 小时，在 60°C 的热应力下保护 30 分钟。这些数据表明了一种通用方法，即将可逆聚合物网络用作复杂生物分子在储存和运输过程中热稳定性的稳健赋形剂，此外还可以在使用点按需释放活性分子。
