Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection

Abstract

Accurate physico-chemical characterization of exosomes and liposomes in biological media is challenging due to the inherent complexity of the sample matrix. An appropriate purification step can significantly reduce matrix interferences, and thus facilitate analysis of such demanding samples. Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) provides online sample purification while simultaneously enabling access to size and Zeta potential of sample constituents in the size range of approx. 1–1000 nm. Hyphenation of EAF4 with Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) detection adds high resolution size and number concentration information turning this setup into a powerful analytical platform for the comprehensive physico-chemical characterization of such challenging samples. We here present EAF4-MALS hyphenated with NTA for the analysis of liposomes and exosomes in complex, biological media. Coupling of the two systems was realized using a flow splitter to deliver the sample at an appropriate flow speed for the NTA measurement. After a proof-of-concept study using polystyrene nanoparticles, the combined setup was successfully applied to analyze liposomes and exosomes spiked into cell culture medium and rabbit serum, respectively. Obtained results highlight the benefits of the EAF4-MALS-NTA platform to study the behavior of these promising drug delivery vesicles under in vivo like conditions.

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers

“…The main challenge is that NTA employs much lower flow-rates than AF4 and therefore cannot be directly coupled to AF4. Several ways to circumvent this problem have been found: to use a switching valve to periodically divert aliquots of the fluid to the NTA chamber [ 73 ], or to split the main stream into multiple flow path by using a splitter manifold connection [ 74 ] or again by using the slot-outlet technology [ 75 ].…”

“…EAF4 was recently used to fractionate liposomal formulations of doxorubicin incubated in phosphate buffer saline (PBS) and Dulbecco’s modified Eagle medium (DMEM) cell culture buffer. The size of the liposomes did not change after incubation in the protein-enriched buffer, whereas their z -potential underwent a 10-mV increase, suggesting that their surface properties changed even if a proper protein corona did not form [ 75 ].…”

Measuring particle concentration of multimodal synthetic reference materials and extracellular vesicles with orthogonal techniques: Who is up to the challenge?

“…Furthermore, the use of a hyphenated technique such as FFF coupled with NTA has recently been presented by Drexel et al. (2020). Also, the adoption of short wavelength lasers and suitable standard controls will allow for more reliable measurements, in particular of small biological nanoparticles with low refractive index.…”

“…A more sophisticated approach for fine-tuning the NTA measurement, avoiding the operator subjective setup, at different size ranges is under development and will be matter for a follow up paper. Furthermore, the use of a hyphenated technique such as FFF coupled with NTA has recently been presented by Drexel et al (2020). Also, the adoption of short wavelength lasers and suitable standard controls will allow for more reliable measurements, in particular of small biological nanoparticles with low refractive index.…”

Dealing with the complexity of conjugated and self‐assembled polymer‐nanostructures using field‐flow fractionation

“…The recent presentation of AF4 hyphenated with nanoparticle tracking analysis (NTA) technology (AF4‐NTA) has brought about efficient and accurate measurements of size distribution and concentration per unit volume for multiple nanoparticles 68 . This presents a wide range of interesting prospects for rapid characterizations of nanostructures in the near future, as NTA not only presents a suitable platform for biomolecule tracking but can also differentiate between molecules of the same sample matrix with different fluorescent markers 69 . The latter attribute is convenient for possibly measuring the degree of functionalization and hybridization for complex and heterogeneous nanostructures.…”