Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles

Abstract

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.

Payload distribution and capacity of mRNA lipid nanoparticles

“…1g, i ), and in quantifying mRNA molecules in mRNA-loaded LNPs. In contrast, fluorescence-based detection may be better suited to elucidating these properties 16 .

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Characterization of nanoparticle size distributions using a microfluidic device with integrated optical microcavities

“…One approach to this would be a configuration in which the trapping laser is 'always on' such that the peak power and average power are equal. Eqn (7) reveals that a factor of 50 increase in P would lead to a concomitant reduction in β T , such that particles about seven times smaller would be trapped. Equivalently a larger quality factor Q ∼ 100 might be achieved by simultaneously increasing the flow speed to around 1.5 mm s −1 to provide a resolution to particle size of below 1 nm or to refractive index of order 10 −3 RIU.…”

“…Therefore, characterizations of nanocarriers such as nanoparticles are crucial to controlling their desired effects 6 . For instance, investigation of the size and loading properties of lipid nanoparticle vehicles containing silencing RNA (siRNA), is of increasing importance for future studies of dynamics with single-particle resolution 7 .…”

“…6 For instance, investigation of the size and loading properties of lipid nanoparticle vehicles containing silencing RNA (siRNA), is of increasing importance for future studies of dynamics with single-particle resolution. 7 There are a number of methods to characterize nanoparticles at the single-particle level with high resolution. Electron microscopy or scanning probe microscopy can provide accurate information on the shape of the nanoparticles.…”

Biophysical Reviews ‘Meet the Editors Series’ — a profile of Sabrina Leslie

“…I am excited to have established active collaborations with academic and private sector partners who helped to pioneer the lipid nanoparticle (LNP) delivery systems that were used in the Pfizer and Moderna mRNA vaccines approved during the COVID-19 pandemic such as Dr. Pieter Cullis (UBC Biochemistry). This collaboration led to our recent publication in ACS Nano of a novel method for simultaneous measurement of LNP size and RNA loading (Kamanzi et al 2021 ). Single-particle imaging of mRNA-LNPs is especially important because the per-particle mRNA-loading can be on the order of unity for standard vaccine formulations, and their structural and dynamic properties are functional but not yet optimized.…”