Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

We directly measure the free energy of confinement for semiflexible polymers from the nanoscale to bulk regimes in slit-like confinement. We use convex lens-induced confinement (CLiC) microscopy of DNA to directly count molecules at equilibrium in a single chamber of smoothly increasing height. Our data, acquired across a continuum of confinement regimes, provide a bridge with which to connect scaling theories established for qualitatively different regimes. We present new experimental data and simulations that connect the Odijk theory describing sub-persistence-length confinement, the interpolation model by Chen and Sullivan extending Odijk to moderate confinement, and the Casassa theory describing the transition from moderate confinement to bulk. Further, this work establishes a robust, quantitative platform for understanding and manipulating biopolymers at the nanoscale, with key applications and insights toward emerging genomic analysis tools.

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