Nanostructured Block Copolymer Gels for Storage and Protection of Concentrated Proteins

用于储存和保护浓缩蛋白质的纳米结构嵌段共聚物凝胶

• 批准号: 1066503
• 负责人: Lynn Walker
• 金额: $ 35万
• 依托单位: Carnegie-Mellon University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-15 至 2014-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1066503&HistoricalAwards=false
• 关键词: Nanostructured; Block; Copolymer; Gels; Storage

AbstractPI: Walker, Lynn M. Proposal Number: CBET-1066503Institution: Carnegie-Mellon UniversityTitle: Nanostructured Block Copolymer Gels for Storage and Protection of Concentrated ProteinsThe PI has addressed the following reviewer concerns:1. The proposed method will require a large amount of polymeric material to create micelles and nanostructured materials. Because purity of protein is generally an important issue to consider for applications involving proteins, the proposed method may require additional purification stages to be an effective strategy for protein storage. The hydrogel-protected protein composite that will result from this approach will have high loadings of protein (~ 30 mg/mL, or 1-3wt%) in a block copolymer matrix that is primarily water (~25-35% polymer and 65-75% water). Many application methods (for example, topical application of proteins that act as antidotes) could be used immediately even at these polymer levels. Since the protein loadings are high, there is room for dilution (with saline or water) on use; this will lower the overall concentration of polymer in the formulation at the point of application. Typical therapeutic concentrations of proteins can be an order of magnitude below the level loaded into the hydrogel, so dilution of the protein will result in a commensurate dilution of the block copolymer. Again, many protein applications will tolerate a small weight percent of polymer in the use. A part of our motivation for choosing Pluronic materials is that these have been FDA approved for several uses; including application to the skin, eyes, oral ingestion and, in some cases, injection. Finally, if complete removal of the polymer is required, it is important to note that the molecular weight of the uncharged block copolymer is lower than most proteins of interest and the hydrodynamic size is considerably smaller. Therefore, either diluted or cooled solutions can be purified with size exclusion or electrokinetic approaches (CE, SEC or even simple dialysis). For highly sensitive proteins, or those that need extremely high levels of purity, the use of nanostructured hydrogels may not be possible. Developing approaches to test whether this approach is effective for a given protein is part of Goals 2 & 3 of the proposed work. For example, light scattering and circular dichroism tests were developed and used to demonstrate that BSA (and lysozyme) can be templated within the hydrogel and recovered. These proteins are not particularly sensitive, but success with these materials demonstrates the strong likelihood for success with a wider range of proteins.2. Also, the proposal does not adequately address issues regarding the protein loading behavior of the proposed nanostructured materials. The loading procedure is part of the novelty of this approach. The thermoreversible hydrogel allows the protein to be dispersed at cold temperatures and then "loaded" into the nanostructured gel simply by warming to temperatures above about room temperature. Proteins are nudged into the interstitial spaces through steric interactions as the block copolymer micelles form. The PI's previous work has demonstrated our ability to use this approach to template nanoparticles and globular proteins (see ref 1-5 and 49 of the proposal). This process is reversible and has been shown (see Fig 4 of proposal) not to cause detrimental effects to the protein, at least at the level of aggregation. Figure 1 of the proposal was intended to show this procedure, but is not as clear as it could have been.3. The advantage of the proposed strategy over the strategies relying on the encapsulation of proteins within liposomes or micelles is not clear.Strategies that focus on encapsulation of proteins in liposomes or micelles are quite different than the proposed approach. Here, the PI is trapping the proteins in the interstitial (water-filled) spaces in the block copolymer crystal, and taking advantage of both the nanoscale confinement and macromolecular crowding mechanisms for protein protection. Other approaches encapsulate the proteins in the cores of reverse micelles, but this requires a continuous solvent phase that is non-aqueous. An advantage of the proposed approach is the use of water (or saline, buffers, etc.) as a solvent rather than non-aqueous solvents which can denature proteins and require a purification step that calls for high levels of purity. Liposomes offer a complimentary technique but are much more complex systems and often involve electrostatics as a force driving self-assembly. Here, the PI avoids this level of complexity, which can lead to specific interactions between the lipids that make up the liposomes and the proteins (specific interactions which can lead to denaturation). Finally, a key to the PI?s approach is the thermoreversibility of the nanostructured hydrogel and the ability to form/break the matrix with small changes in a temperature range that does not damage proteins. This structural control is not available in micelle or liposome encapsulation.

摘要：Walker，Lynn M.提案编号：CBET-1066503Institution：Carnegie-Mellon Universitytitle：用于储存和保护浓缩蛋白的纳米结构块共聚物凝胶PI已解决了以下审阅者的关注者：1。提出的方法将需要大量的聚合物材料来产生胶束和纳米结构材料。由于蛋白质的纯度通常是要考虑涉及蛋白质的应用的重要问题，因此所提出的方法可能需要额外的纯化阶段才能成为蛋白质储存的有效策略。该方法将产生的水凝胶保护的蛋白质复合材料将具有高负载的蛋白质（〜30 mg/ml，或1-3wt％），该蛋白主要是水（主要是〜25-35％聚合物和65-75％的水）。 即使在这些聚合物水平上，许多应用方法（例如，用作解毒剂的蛋白质的局部应用）也可以立即使用。 由于蛋白质负荷很高，因此使用稀释的空间（盐水或水）。这将降低在施用点的配方中聚合物的总体浓度。 蛋白质的典型治疗浓度可以比负载到水凝胶的水平以下的数量级，因此蛋白质的稀释将导致块共聚物的相应稀释。 同样，许多蛋白质的应用将忍受使用中聚合物的一小部分。 我们选择复数材料的动机的一部分是，这些材料已被FDA批准用于多种用途。包括对皮肤，眼睛，口腔摄入以及在某些情况下注射的应用。 最后，如果需要完全去除聚合物，重要的是要注意，未加成的块共聚物的分子量低于大多数感兴趣的蛋白质，并且流体动力学大小要小得多。 因此，可以使用尺寸排除或电动方法（CE，SEC甚至简单的透析）纯化稀释或冷却的溶液。 对于高度敏感的蛋白质或需要极高纯度的蛋白质，可能无法使用纳米结构水凝胶。 开发测试这种方法是否对给定蛋白有效的方法是拟议工作的目标2和3的一部分。 例如，开发了光散射和圆形二分性测试，并用于证明BSA（和溶菌酶）可以在水凝胶中模板并回收。 这些蛋白质并不是特别敏感，但是这些材料的成功表明，通过蛋白质范围更广泛，成功的可能性很大。2。同样，该提案不能充分解决有关拟议纳米结构材料的蛋白质负荷行为的问题。加载过程是这种方法的新颖性的一部分。 可爱的水凝胶使蛋白质可以在寒冷的温度下分散，然后仅通过温暖到高于室温的温度来“加载”到纳米结构的凝胶中。 蛋白质通过空间相互作用将蛋白推入间质空间中，因为块共聚物胶束形成。 PI先前的工作证明了我们使用这种方法将这种方法用于模板纳米颗粒和球形蛋白（请参阅提案的参考文献1-5和49）。这个过程是可逆的，并且已显示（请参见提案的图4），至少在聚集水平上不会对蛋白质造成不利影响。 该提案的图1旨在显示此程序，但并不像以前那么清楚。3。拟议策略比依赖于脂质体或胶束中蛋白质封装的策略的优势尚不清楚。专注于在脂质体或胶束中封装蛋白质的策略与建议的方法完全不同。 在这里，PI将蛋白质捕获在块共聚物晶体中的间隙（水填充）空间中，并利用纳米级限制和大分子分子拥挤机制来保护蛋白质保护。 其他方法将蛋白质封装在反向胶束的核心中，但这需要一个无水的连续溶剂相。 提出方法的一个优点是将水（或盐水，缓冲液等）用作溶剂而不是非水的溶剂，该溶剂可以使蛋白质变质，并且需要一个需要高水平纯度的纯化步骤。 脂质体提供了一种免费的技术，但系统更为复杂，并且通常涉及静电作为驱动自组装的力。 在这里，PI避免了这种复杂性，这可能导致构成脂质体和蛋白质的脂质之间的特定相互作用（可能导致变性的特定相互作用）。 最后，PI方法的关键是纳米结构水凝胶的热可逆性，以及在温度范围内不损害蛋白质的温度范围内形成/破坏基质的能力。 这种结构控制在胶束或脂质体封装中不可用。