Objectives
This project has two thrusts: The first thrust (and the main focus of our continuation project) combines X-shaped DNA with a thermal reversible polymer (pNIPAAm) to create a gel that can expand or shrink in response to a small change in temperature. After endocytotic delivery to a cell at 37°C and lowering the temperature briefly to 32°C, the Spongel expands to sample intracellular molecules in situ by absorption. The Spongel can then be isolated from the cell (by exocytosis or cell lysis), and raising the temperature to 37°C causes the intracellular contents trapped inside to be expelled for further analysis. Similarly, by encapsulating probes and drugs inside our Spongel, we can deliver these intracellularly by altering the temperatures. Thus we will be able to sample, analyze, and map molecular compositions and compositional changes within intracellular vesicular compartments. We intend to develop this enabling technology for investigating membrane trafficking and molecular constituents involved in antigen-stimulated, receptor-mediated cell activation, particularly endosomal and secretory pathways. The second thrust extends our previous success using branched DNA as a scaffold for IgE-specific ligands of defined lengths that distinctively activate IgE receptor signaling in mast cells. We are using scanning electron microscopy (SEM) to evaluate nanoscale clustering of IgE-receptors and related signaling components after crosslinking with Y-shaped DNA ligands, and we are developing gold-labeled DNA-ligands for direct visualization.
Methods
Development of Spongels for cell biology studies (Thrust 1) involves: 1) Spongel synthesis (Luo) 2) Spongel characterization (Luo and Baird), and 3) Analysis of cellular contents and connecting to cell biology (Baird). Synthetic methods include nanoscale, branched DNA structures and DNA-pNIPAAm hybrid molecules. Degree of crosslinking is varied to change the lower critical solution temperature (LCST). Microfluidic devices are used to generate nano-droplets that can be endocytosed inside cells, with or without targeting to specific receptors. Characterization methods include SEM and fluorescence microscopy to evaluate cellular localization as well as contraction and expanding behaviors of Spongels under different cues (e.g., temperatures, pH, etc.). We will evaluate release dynamics of model dyes in vitro and in vivo. Spongel localization and molecular uptake will be related to endosomal pathways and known protein markers that may be altered under conditions of cell activation. Isolated Spongel absorbates will be evaluated with ELISAs, Western Blots, and possibly mass spectrometry to follow changes in membrane trafficking corresponding to signal transduction. For Thrust 2, Y-shaped DNP-DNA ligands of different lengths are used to crosslink anti-DNP IgE receptors (gold labeled) on cells that are visualized with SEM. Degree of clustering is quantified with correlation analysis.
Summary
We successfully fabricated Spongels in the nanometer sizes (800 nm based on dynamic light scattering and SEM) and initiated efforts with DNA-pNIPAAm hybrid gels. We find Spongels absorb a model dye, Texas-Red-conjugated Dextran. In vitro release of dye and in vivo endocytosis by cells are being characterized. Fabricated microfluidic devices for generating gel-droplets provides a micro-meter sized environment for nano-spongel formation. New multivalent, anisotropic DNA monomers that can photo-crosslinked into polymers will facilitate spongel formation as a platform technology. Soluble ligands based on dendrimeric DNA scaffolds are proving valuable for SEM analysis of activated cell surface receptors and intracellular signaling components that co-redistribute.
Accomplishments
- Prepared Spongels that are less than 1 µm and absorb model drug
- Developed microfluidic devices that can generate droplets in the gel format
- Quantified IgE clustered with DNA ligands on cells using SEM and correlation analysis
