The 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.
The motivation for this work arises from the observation that nanostructured block copolymer solutions and gels are able to trap and hinder the motion of nanoparticulate material. This provides the potential for trapping globular proteins in these water-based solutions and gels. Trapped, or templated, proteins should be protected from aggregation and denaturation, two mechanisms that can lead to loss of efficacy in protein-based drugs. The goals of the work were to 1) Characterize the structure and diffusion in gel-protein systems; (2) Quantify the ability of nanostructured hydrogels to hinder aggregation of target proteins; and (3) Determine the ability of nanostructured hydrogels to hinder thermal denaturation near and above the denaturation temperature. All three goals were met. Diffusion of a globular protein, bovine serum albumin, was quantified in a series of block copolymer systems and the mechanisms of transport or diffusion in the matrices determined. Aggregation of both proteins and inorganic nanoparticles was characterized and the impact of the details of the interfacial properties of these on aggregation determined. Results of the first parts of the project provided rules to design block copolymer systems to optimize protection of proteins. Protection of proteins against aggregation was observed and some limited protection from thermal denaturation found, although the nanostructured systems may not provide the confinement necessary to stop all thermal denaturation. The broader impact of this work is that these nanostructured materials have been shown to have the potential to provide protection for proteins during storage and transport. Three graduate students and one undergraduate student obtained training in research as part of this work. Outreach to larger groups of K-12 students provided exposure to the exciting field of soft materials.
