Dabas Rupali, Blagojevic Luka, Kamaly Nazila
Department of Chemistry, Molecular Sciences Research Hub, Imperial College London.
Department of Chemistry, Molecular Sciences Research Hub, Imperial College London;
J Vis Exp. 2025 Jan 24(215). doi: 10.3791/63981.
Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N',N'-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.
由交联聚合物纳米颗粒组成的纳米凝胶已被开发用于递送多种化学和生物治疗剂，这得益于其多样的自下而上合成方法和生物相容性。尽管迄今为止已采用各种方法来合成纳米凝胶，但很少有方法能在不使用可能损害生物药物完整性的苛刻有机溶剂或高温的情况下实现合成。相比之下，本文介绍的方法在温和的反应条件下完成了尺寸小于100 nm的载蛋白纳米凝胶的合成。在此，我们提出了一种将基于蛋白质的药物非共价包封在纳米凝胶中的方法，该纳米凝胶是使用水相单步交联共聚技术合成的。在这项技术中，我们首先将基于蛋白质的药物静电结合到阳离子季铵单体上，同时使用过硫酸铵和N,N,N',N'-四甲基乙二胺对其进行交联和共聚，以形成包裹蛋白质药物的纳米凝胶。使用动态光散射（DLS）测定纳米凝胶的尺寸和多分散指数，同时通过透射电子显微镜（TEM）评估其表面形态。通过计算包封效率来确定纳米凝胶中包裹的蛋白质质量。此外，还在生物还原试验中评估了纳米凝胶通过氧化还原响应结构元件的逐渐降解实现的控释能力。我们提供了纳米颗粒优化数据的示例，以证明使用该技术进行纳米凝胶合成和表征的所有注意事项。一般来说，获得了尺寸均匀的纳米凝胶，平均尺寸为57 nm，多分散指数值为0.093。实现了76%的高包封效率。此外，在谷胱甘肽存在的情况下，通过新型氧化还原响应成分在48小时内的逐渐降解，纳米凝胶显示出高达86%的包裹蛋白控释率。
