Application Guide: Lipid Nanoparticles (LNPs)

Spectradyne’s ARC™ Particle Analyzer
for Lipid Nanoparticle Characterization

Published:
Revision: V3

Overview

Spectradyne’s ARC™ particle analyzer is rapidly being adopted in QC and extended characterization workflows for fast and complete lipid nanoparticle (LNP) characterization. The ARC provides more direct and accurate measurements of LNP size, concentration, and loading than purely optical methods.

From simple measurements of nanoparticle size and concentration to in-depth studies of targeting ligand density and co-encapsulation of multiple payloads, the ARC delivers fast, reliable, and easy-to-use measurements that deepen formulation understanding and accelerate development.

How the ARC Particle Analyzer Works

Spectradyne’s ARC particle analyzer uniquely combines two orthogonal measurement techniques to yield new insights into LNP formulations.

  1. Microfluidic Resistive Pulse Sensing (MRPS) directly measures single-particle size and concentration. MRPS is a non-optical, electrical technique that is orthogonal to Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Flow Cytometry (FC).
  2. Quantitative single-particle fluorescence directly quantifies encapsulated payload. Color multiplexing enables simultaneous measurement of multiple payloads, co-encapsulation, and targeting ligand density.

Gold-Standard Accuracy for Size and Concentration

Comparison of LNP size and concentration measurements using MRPS and Cryo-TEM
Figure 1. Direct comparison of size and concentration measurements for an mRNA-loaded LNP formulation using MRPS and Cryo-TEM. In approximately two minutes, the ARC measured 1906 particles, yielding a concentration of 4.2 × 10¹³ particles/mL across the measured size range.

MRPS and Cryo-TEM show remarkable agreement across the full measurement range, validating the ARC’s accuracy for LNP size and concentration analysis.

Payload Quantification (Empty / Full)

In addition to size and concentration, the ARC measures payload encapsulation on a single-particle basis. A ten-minute, no-wash staining protocol enables internal payload quantification directly as a function of particle size.

Fluorescence intensity is reported in NIST-defined units of equivalent reference fluorophore (ERF), enabling conversion to absolute numbers of molecules per particle.

Separation of empty and payload-loaded LNPs using single-particle fluorescence Scatter plot of separation of empty and payload-loaded LNPs using single-particle fluorescence
Figure 2. Payload quantification of an mRNA-loaded LNP formulation. Size distributions of all particles (blue) and fluorescently loaded particles (green) are shown. Approximately 62% of particles within the measured size range are payload-loaded.

Quantifying Targeting Ligands

Functionalization of LNPs with targeting ligands is a critical development frontier. The degree of functionalization is a key quality attribute for assessing formulation performance and interpreting downstream potency assays.

In this example, LNPs incorporating Alexa Fluor 647–conjugated surface ligands were measured using red-laser excitation on the ARC particle analyzer.

Fluorescence-based quantification of targeting ligands on LNP surfaces Fluorescence-based quantification of targeting ligands on LNP surfaces, scatter plot
Figure 3. Fluorescence analysis of ligand-functionalized LNPs. The particle size distribution shows a mean diameter of 83.3 nm with a CV of 19.6% and a total concentration of 1.1 × 10¹⁰ particles/mL. Ligand-positive particles represent 98.3% of the measured population.

Co-Encapsulation of Multiple Payloads

As therapeutic LNP applications expand, formulations increasingly require co-encapsulation of multiple nucleic acid payloads. The relative fractions of empty, single-payload, and dual-payload particles are critical quality attributes.

Using fluorescence multiplexing, the ARC particle analyzer quantifies co-encapsulation on a particle-by-particle basis in just a few minutes.

Quadrant analysis showing co-encapsulation of two nucleic acid payloads in LNPs
Figure 4. Co-encapsulation analysis of two nucleic acid payloads labeled with distinct fluorochromes. Quadrant analysis shows that approximately 44% of particles contain both payloads.