Friday, April 4, 2025
4/4/2025
Nanopore Force Spectroscopy and Sorting of Vesicles at Nanoscale - PROJECT SUMMARY Soft nanoparticles like exosomes, liposomes and viruses play a vital role in biological and physiological (including therapeutic) functions such as exo- and endocytosis, membrane trafficking, and intercellular signaling. With advancements in targeted drug/gene delivery, cargo carrying vesicles with minimal systemic toxicity, improved uptake and controlled drug release at the cellular/tissue targets have gained substantial attention. A key functional aspect of these soft vesicles is the ability to deform relative to the cargo content, membrane composition and inner/outer media to fuse with cellular organelles and pass through narrow pores. Thus, profiling deformability is critical for understanding their functions and successful drug engineering. The existing classical techniques like atomic force microscopy (AFM) demand laborious and cumbersome procedures, with low throughput. Other available methods to study the cargo encapsulated by the vesicles such as DNase digestion, fluorescence, and UV absorbance suffer from overnight procedures, DNA extraction (and tagging), and DNA standard curve requirements, respectively. In this proposed work, we intend to develop a technique to overcome all these shortcomings to study the deformability of soft nanoparticles by using a nanopore – a nanoscale channel separating two electrolyte chambers through which analyte particles can electrokinetically travel from one chamber to the other in response to an applied bias, one at a time, causing electrical perturbations unique to particle size, shape, and content. Nanopores are robust, sensitive and inexpensive miniature sensors of higher throughput – capable of studying thousands of particles within seconds to minutes. We will first develop a computational model(s) for electro-deformability of the liposomes which depends on conductivity, cargo density, and surface charge (Aim 1a). We propose an automated recapture protocol to study the same particle ~25 times by reversing the voltage bias after each translocation event to re-translocate the same particle to generate a large pool of reliable statistics on deformability of the liposomes. Electro- deformability would be expressed as the relative current drop ratio at forward and reverse biases. The obtained results will be compared with a commercially available, incompressible rigid particle like polystyrene to accredit the results (Aim 1b). It is assumed that the cargo content would affect the deformability of the liposomes by altering the inner solution conductivity and perhaps the shape. We propose to synthesize nanoliposomes housing ss/ds-DNA of known ratios to test this theory (Aim 2a). By studying the electro-deformability of DNA- encapsulated liposomes, in comparison with cargo-free liposomes, we intent to determine the degree of electro- deformability in response to the cargo content (amount and percentage) (Aim 2b). In addition, the automated recapture protocol will enable the discrimination of samples of desired mechanical properties out of a population of vesicles with varied properties. Once parallelized, high throughput data can be obtained to characterize rigidity of any nanoscale soft particle at the single-particle level.
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