UC San Diego
Polymer and Nucleic Acid Self-Assemblies: Properties and Applications at the Biological Interface
- Author(s): Barnhill, Sarah Anne
- Advisor(s): Gianneschi, Nathan C
- et al.
Ring opening metathesis polymerization (ROMP) was used to generate a variety of self-assembled nanostructures, including purely synthetic and bio-hybrid materials. The properties of polynorbornene amphiphilic block copolymer structures and their relationship to resulting morphology was explored, paving the way for nanoparticle design at the structural and processing level. In the context of bio-synthetic polymer amphiphiles, hydrophobic ROMP polymers were attached to hydrophilic DNA strands to produce self-assembly micelles on the order of 20 nm in diameter. Herein, we explored the self-assembly properties, stability, and applications of these assemblies in pristine conditions and cellular environments.
Morphology plays an important yet poorly understood role in dictating how nanomaterials interact with cells and tissues. Uncovering this relationship relies on working out how to control particle morphology in the first place. We prepared purely synthetic amphiphiles using ROMP to prepare aqueous phase diagrams of block copolymer assemblies. By preparing polymers with varying properties, such as block lengths, block identity, and block ratios, the relationship between polymer structure and the resulting self-assembly nanostructure could be observed under certain conditions. Furthermore, by manipulating the assembly conditions of these polymers, we have shown that multiple stable morphologies can be generated from the same block copolymer starting material. This represents the first study of its kind for ROMP-derived amphiphilic assemblies, which exhibit variations in self-assembly dynamics compared to more traditional block copolymers.
Adding a level of complexity to our block copolymer system, we next explored more therapeutically relevant systems by conjugating DNA to a hydrophobic ROMP homopolymer and assembling them into DNA-displaying micelles. We determined the stability of the DNA on the micelle surface by treating the structures with various nucleases and human serum. The stability of the DNA on the micelle corona resisted degradation by nucleases in some circumstances, but not all, relative to the free DNA control, highlighting the importance of careful design of the amphiphile for a given application.
After determining the stability of DNA polymer assemblies (DPAs), antisense DPAs were designed against a known therapeutic target in cancer cells, MDR1. Importantly, these materials were designed with sequences not containing chemical modifications, such as locked nucleic acids or other backbone alterations. After treating MDR1-dependent doxorubicin resistant cells with the antisense micelles, sensitivity to the chemotherapeutic could be restored to near-parental cell line IC50 values.
Despite many desirable properties nanoparticles have in therapeutic applications, a major bottleneck in their development is the fact that very little is known about how they interact with cells and tissues. Next, a high throughput, whole-genome approach to elucidate the pathways responsible for nanomaterial uptake by cells was developed and tested using DPAs as a proof-of-concept. Using Genome-wide CRISPR Knock Out (GeCKO), a population of cells representing knockouts across the entire genetic spectrum was tested against uptake of cyanine 5 labeled DPAs. Using this approach, we have identified the transmembrane protein SLC18B1, among a handful of other proteins, as candidates for mediating uptake; a previously unknown interaction by DNA-displaying nanomaterials with cell surfaces. By expanding this technique to other categories of nanoparticle medicines with different structures and surface modifications the generation of new design rules for nanomaterial therapeutics may be prepared to help researchers avoid off-target accumulation and advance many more nanotherapies to the clinic.