Nanomedicine, the application of nanotechnology in medical fields, has made significant strides, particularly after the COVID-19 pandemic, where lipid-based nanoparticles became the favored carriers for genetic material. These advancements have highlighted the importance of optimizing nanoparticles for safety and clinical application. This requires thorough characterization of critical quality attributes (CQAs) such as particle size, particle size distribution (PSD), shape, and surface charge. Each characterization technique has its own limitations, necessitating a combination of orthogonal (different physical principles) and complementary (same physical principles) methods to achieve a comprehensive understanding.

Characterization Techniques Overview

Dynamic Light Scattering (DLS)

• Pros: Quick, non-invasive, cost-effective.
• Cons: Sensitive to polydisperse samples; large particles can obscure smaller ones.
• Measures: Hydrodynamic diameter (Z-average), Polydispersity Index (PDI), Zeta potential.
• Detection Angle: Commonly 90°, but backscattering at 173° is useful for turbid samples.

Nanoparticle Tracking Analysis (NTA)

• Pros: Real-time tracking, detailed size distribution.
• Cons: Requires high sample volume (but lower concentration).
• Measures: Number of particles per mL, Hydrodynamic radius.
• Range: 30 – 1000 nm.

Asymmetric Flow Field-Flow Fractionation (AF4) with Multi-Angle Light Scattering (MALS)

• Pros: High-resolution size separation, coupled with DLS.
• Cons: Complex, requires skilled operators.
• Measures: Particle size distribution (PSD), Molecular weight distribution.
• Standards: ISO/TS 21362:2018.

Transmission Electron Microscopy (TEM)

• Pros: High-resolution imaging of particle size and shape.
• Cons: Labor-intensive sample preparation, potential artifacts.
• Measures: Particle size, Shape, Morphology.

Analytical Ultracentrifugation (AUC)

• Pros: High-resolution size distribution, true orthogonal technique.
• Cons: Expensive equipment, requires expertise.
• Measures: Sedimentation coefficient, Particle size distribution.

Small-Angle X-ray Scattering (SAXS)

• Pros: Detailed structural information.
• Cons: Requires synchrotron facilities.
• Measures: Structural information, Particle size, Shape.

Combining Techniques

Using a mix of these techniques allows us to cross-verify results and gain a more objective understanding of the CQAs of nanomedicines. For instance, while DLS provides a quick prescreening, TEM offers detailed insights into particle morphology. Integrating AF4-MALS-DLS provides high-resolution data crucial for regulatory standards.

Conclusion

Thorough characterization of nanomedicines using diverse techniques is essential for advancing their clinical applications. By understanding the strengths and limitations of each method, researchers can better optimize nanoparticle formulations for safety and efficacy.

How to calculate volume, mass and conc of particles https://nanocomposix.com/pages/nanoparticle-volume-mass-and-concentration

EU NCL Assay cascade https://www.euncl.org/about-us/assay-cascade/

US NCL Assay cascade https://www.cancer.gov/nano/research/ncl/protocols-capabilities

Written by

Dr. Marija Petrovic 

Marija is a pharmacist with a PhD in Biopharmacy from the University of Geneva, and a cancer research (ISREC)–trained professional through EPFL, with over seven years of experience in nanomedicine. During her PhD, she worked on miRNA and STING ligand nanocomplexes for cancer immunotherapy, gaining deep expertise in nanoparticle characterization and translational workflows. Certified by the EU-NCL in nanobiotechnology and awarded by Innosuisse (Swiss Innovation Agency) with two prizes (jury and public) for the best life science project, she also earned support from FONGIT, Geneva’s leading deep-tech incubator. As the founder of NanoSphere and an active contributor to the Controlled Release Society (Communication Chair for the Gene Delivery and Editing Group (GDGE), and Industry representative at Nanomedicine and Nanoscale Delivery (NND)), Marija focuses on making next-gen medicine scientific advances more visible, understandable, and useful to the communities that can turn them into impact.