Nanostructured Block Copolymer Gels for Storage and Protection of Concentrated Proteins

摘要PI: Walker， Lynn M.提案编号：cbet -1066503机构：卡内基梅隆大学标题：用于储存和保护浓缩蛋白质的纳米结构嵌段共聚物凝胶PI解决了以下审稿人关注的问题：所提出的方法将需要大量的聚合材料来制造胶束和纳米结构材料。由于蛋白质的纯度通常是涉及蛋白质的应用中需要考虑的一个重要问题，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。通过这种方法得到的水凝胶保护的蛋白质复合材料将在主要为水的嵌段共聚物基体中具有高负载的蛋白质（~ 30 mg/mL，或1-3wt%）（~25-35%的聚合物和65-75%的水）。许多应用方法（例如，局部应用作为解毒剂的蛋白质）即使在这些聚合物水平上也可以立即使用。由于蛋白质含量高，使用时有稀释的余地（用生理盐水或水）；这将降低在应用时配方中聚合物的总浓度。典型的治疗浓度的蛋白质可以是一个数量级以下的水平加载到水凝胶，因此蛋白质的稀释将导致相应的嵌段共聚物的稀释。同样，许多蛋白质应用将在使用中容忍一小部分重量百分比的聚合物。我们选择Pluronic材料的部分动机是，这些材料已被FDA批准用于多种用途；包括皮肤、眼睛、口服和在某些情况下注射。最后，如果需要完全去除聚合物，重要的是要注意，不带电的嵌段共聚物的分子量比大多数感兴趣的蛋白质要低，而且水动力尺寸要小得多。因此，稀释或冷却的溶液都可以通过尺寸排除或电动方法（CE， SEC甚至简单的透析）进行纯化。对于高度敏感的蛋白质，或者那些需要极高纯度的蛋白质，使用纳米结构的水凝胶可能是不可能的。开发测试该方法对特定蛋白质是否有效的方法是拟议工作的目标2和目标3的一部分。例如，开发了光散射和圆二色性测试，并用于证明牛血清白蛋白（和溶菌酶）可以在水凝胶中模板化并回收。这些蛋白质并不是特别敏感，但这些材料的成功证明了在更广泛的蛋白质上取得成功的巨大可能性。此外，该提案没有充分解决有关所提议的纳米结构材料的蛋白质负载行为的问题。加载过程是这种方法新颖之处的一部分。热可逆性水凝胶允许蛋白质在低温下分散，然后通过加热到室温以上的温度“装载”到纳米结构凝胶中。当嵌段共聚物胶束形成时，蛋白质通过空间相互作用被推入间隙。PI之前的工作已经证明了我们使用这种方法来模板纳米颗粒和球状蛋白的能力（见提案的参考文献1-5和49）。这个过程是可逆的，并且已经证明（见提案的图4）不会对蛋白质造成有害影响，至少在聚集水平上是这样。该提案的图1旨在显示这一程序，但并不像它本来可以的那样清楚。与依赖于脂质体或胶束内的蛋白质封装的策略相比，所提出的策略的优势尚不清楚。将蛋白质包封在脂质体或胶束中的策略与提出的方法完全不同。在这里，PI将蛋白质捕获在嵌段共聚物晶体的间隙（充满水）空间中，并利用纳米级限制和大分子拥挤机制来保护蛋白质。其他方法将蛋白质包裹在反胶束的核心中，但这需要非水的连续溶剂相。该方法的一个优点是使用水（或盐水、缓冲液等）作为溶剂，而不是非水溶剂，因为非水溶剂会使蛋白质变性，并且需要一个要求高纯度的纯化步骤。脂质体提供了一种互补的技术，但它是更复杂的系统，通常涉及静电作为驱动自组装的力。在这里，PI避免了这种复杂程度，这可能导致构成脂质体的脂质与蛋白质之间的特定相互作用（可能导致变性的特定相互作用）。终于找到PI的钥匙了？S方法是纳米结构水凝胶的热可逆性，以及在不损害蛋白质的温度范围内通过微小变化形成/破坏基质的能力。这种结构控制在胶束或脂质体封装中是不可用的。

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.