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Molecular Evolution of the Primary Structure of Single Chain Polymer Nanoparticles via Dynamic Covalent Chemistry

Periodic Reporting for period 1 - MolecularEVOLUTION (Molecular Evolution of the Primary Structure of Single Chain Polymer Nanoparticles via Dynamic Covalent Chemistry)

Reporting period: 2016-03-01 to 2018-02-28

Despite immense resources dedicated to its treatment, cancer remains one of the most common causes of death worldwide. A key problem with chemotherapy, the most common form of treatment, is that it does not discriminate between normal and cancerous cells, causing serious side effects. A promising strategy to overcome this issue is to utilize a synthetic material that can activate a chemotherapy pre-drug at the site of the tumor—in this way, the drug will remain inactive until it reaches the desired site in the body and result in fewer side effects. What kind of material could achieve this function? We take inspiration from enzymes, which are specialized proteins that act as highly active and selective catalysts in cells. A key contributor to their performance is the precise folding of polypeptide chains around the enzyme’s active site. Possible conformations of the polypeptide chains are fundamentally determined by the primary structure of the polypeptide, the sequence of amino acids. In our research, we are interested in exploiting amphiphilic polymers with pendant “sticky” hydrogen bonding moieties and catalytic centers for targeted drug delivery therapies. When these polymers are dissolved in dilute aqueous solutions, individual chains fold to form single chain polymer nanoparticles (SCPNs), resembling “synthetic enzymes.” However, in contrast to the well-defined structure of natural enzymes, current SCPNs exhibit open, elliptical structures. We proposed that this lack of high-order structure is due to current SCPNs having random sequences and theorized that the activity of SCPNs could be enhanced by optimizing the sequence.

The primary objective of this project was to develop new fundamental chemistry to control the high order structure of SCPNs. We sought to design SCPNs with reversible connections so that the sequence could be “shuffled” in conjunction with the dynamic aggregation of the “sticky” pendant units to “molecularly evolve” the SCPN primary structure. This strategy allows for individual SCPNs to thermodynamically optimize their structure to achieve a lower energy state, leading to a better defined core for performing catalytic reactions. We have developed new chemistry that allows us to “shuffle” the sequences of SCPNs and track the changes of this process by nuclear magnetic resonance spectroscopy. Additionally, in response to challenges in achieving later project milestones, we pursued an alternative line of research to improve SCPN folding. We investigated “stickier” aggregation units, envisioning that they could be used to more efficiently fold SCPNs, and discovered a new class of “stickier” units that form one-dimensional stacks. Remarkably, these stacks change their helicity as a function of temperature, and we ultimately discovered that these transitions are caused by water molecules binding to the stacks. A manuscript that describes these discoveries was recently accepted for publication in Nature and give researchers better insight into fundamentals of hydrophobic effects. We believe that this technology will be key in developing SCPNs with better folding and temperature responsiveness.
This project consisted of two main parts: 1) development of technology for “shuffling” the sequences of polymers that form single chain polymer nanoparticles (SCPNs) and 2) exploring new “stickier” units for driving SCPN folding. First, we aimed to synthesize polycarbonate-based SCPNs that could undergo shuffling through transesterification chemistry. We quickly found that this approach was not feasible in aqueous media, leading to hydrolysis of the polymer. As outlined in our risk assessment plan, we thus sought to develop proof-of-concepts in organic solvent. Another important issue we encountered was a lack of analytical methods available for answering basic questions about the shuffling process. For example, in a transesterification-based shuffling experiment, it was clear that shuffling occurs based on a change in the molecular weight of the polymer, but we could not glean insight regarding the positions of the shuffled units based on standard analytical methods. To solve this issue, we decided to shift focus on Diels-Alder-based shuffling. This was an effective choice, as we found that substituents connected to the main backbone could be shuffled through thermal control. Furthermore, by strategically deuterium-labelling these connections, we could track shuffling through a combination of proton and deuterium nuclear magnetic resonance microscopy. To finish this project for publication, research now is focused on quantitatively establishing the relationship between shuffling behavior and the aggregation of the “sticky” hydrogen bond-based units. This work was orally presented at the 2016 American Chemical Society National Meeting 2016 in Philadelphia (USA).

In the second component of this project, we aimed to explore new hydrogen bond-based “sticky” groups used to fold polymers into SCPNs; we envisioned that the efficiency of the folding could be increased by using a “stickier” unit. We targeted a biphenyl tetracarboxamide (BPTA), predicting that it would be stronger. Our experiments with a model BPTA compound confirmed that these units bind more strongly than our older design, forming one-dimensional helical fibers. Intriguingly, these fibers change their helicity as a function of temperature; we ultimately discovered that the binding of water molecules is responsible for this change in structure. These insights are unprecedented in the field of supramolecular chemistry and resulted in a manuscript that was recently accepted for publication in Nature. This project has also been orally presented at the 2016 and 2017 American Chemical Society National Meetings as well as 2016 CHAINS and the 2018 Dutch Polymer Days. As soon as the Nature manuscript is published online, we will further promote this work through a university news article, social media announcements, and a blog post on the Nature Research Chemistry Community website. Additionally, as part of a collaboration with the Voets group at the Eindhoven University of Technology, we developed a new super-resolution fluorescence microscopy technique for visualizing these fibers. A manuscript detailing this technique was just accepted for publication in ACS Nano.
Improving public health remains a core goal of our research efforts, and we believe that advances toward next-generation targeted drug delivery therapies will require advances in fundamental science. The technology generated in this project serve as an exciting step toward developing new polymeric nanoparticles that are able to achieve higher-order structure and potentially higher activities as bio-orthogonal catalysts. As this project reaches a close, we are currently preparing a manuscript that will disclose the final results of our shuffling experiments. Future efforts in this field will be based on incorporating our technology into water-soluble prototypes and incorporating catalysts into them. Furthermore, the insights reported in our recent manuscripts in Nature and ACS Nano describe a new “stickier” hydrogen bond-based unit. Integrating this design into SCPNs is anticipated to improve folding efficiency. Moreover, the fundamental insight into using water as a structural unit in self-assembly is an exciting starting point for developing new temperature-responsive polymeric devices.
Molecular evolution cartoon