Characterizing Dendrimers by Asymmetric Flow Field Flow Fractionation

Importance of the Topic

Dendrimers are highly branched synthetic macromolecules with well‐defined size, shape and narrow molar mass distribution. Their versatile surface chemistries make them ideal for applications in drug delivery, gene transfection and nanotechnology. Accurate characterization of their size, molar mass and aggregation state is essential to ensure reproducibility and performance in pharmaceutical and biomedical applications.

Objectives and Study Overview

This work evaluates the use of Asymmetric Flow Field Flow Fractionation (AF4) coupled with multiangle light scattering (MALS) and dynamic light scattering (DLS) to characterize generations 4 through 7 of amine‐terminated PAMAM dendrimers. The goals are to assess molar mass, detect fragments and aggregates, compare experimental values against theoretical manufacturer data, and demonstrate the advantages of AF4 over traditional chromatographic methods.

Methodology and Instrumentation

• Separation: AF4 with a 5 kDa regenerated cellulose membrane and long channel format, constant cross flow of 3.0 mL/min for 20 minutes, channel flow 0.6 mL/min.
• Detection: Eclipse Dualtec AF4 system coupled to a Heleos II MALS detector and TrEX refractive index detector; online QELS (DLS) at detector position 16.
• Mobile phase: Phosphate-buffered saline (50 mM phosphate, 50 mM NaCl) to maintain physiological conditions and avoid nonspecific interactions.
• Batch DLS: Supplemental measurements on filtered and centrifuged samples to confirm hydrodynamic radii and aggregation behavior.

Main Results and Discussion

• AF4-MALS separation resolved monomeric dendrimer populations from low‐molecular‐weight fragments and high‐molecular‐weight aggregates in a single run.
• Measured molar masses for G4–G7 were consistently slightly lower than theoretical values, indicating possible polymerization defects and incomplete branching.
• Fragment and aggregate fractions increased with generation number, with mass fractions of fragments rising from under 5 % for G4 to over 10 % for G7.
• Hydrodynamic radii from AF4-DLS aligned closely with theoretical radii for lower generations but revealed unexpected associations in G5–G7 consistent with batch DLS findings.
• Injection volume variation confirmed method robustness and reproducibility of molar mass distribution profiles across differing sample loads.

Benefits and Practical Applications

• AF4 provides a gentle, stationary-phase‐free separation that preserves native dendrimer structures and noncovalent assemblies.
• Ability to use physiological buffers simplifies sample preparation and downstream biological testing.
• Coupling AF4 with MALS and DLS delivers absolute molar mass, size and aggregation state information in a single analysis.
• Method is broadly applicable to quality control of dendrimer‐based formulations, nanomedicine development and polymer manufacturing.

Future Trends and Opportunities

Further integration of AF4 with advanced detectors such as multiangle fluorescence or coupling to mass spectrometry could deepen structural insights. Expanding the technique to other nanoparticle platforms, including lipid‐ or peptide‐based carriers, will broaden its impact in nanopharmaceutical quality assurance. Automation of AF4 protocols and data analysis pipelines can enhance throughput for industrial and regulatory settings.

Conclusion

Asymmetric Flow Field Flow Fractionation coupled with light scattering detectors offers a comprehensive, gentle and high‐resolution approach for characterizing PAMAM dendrimers. It reliably quantifies molar mass, size and aggregation states across multiple generations, outperforming conventional SEC in compatibility with physiological buffers. This method is a powerful tool for research, development and quality control of dendrimer‐based nanomedicines.