Field-Flow Fractionation

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With many years of custom mRNA delivery research experience, Creative Biolabs prides itself on being an innovator and problem solver in the mRNA delivery industry. With the facility, knowledge, and technology that Creative Biolabs possesses, we can offer custom solutions in a variety of mRNA delivery. Asymmetric-flow field-flow fractionation (AF4) is a very powerful technique for the analysis of particle size, polydispersity, and physical stability of nanopharmaceuticals (NPhs) in complex biological media, and also for assessing particle concentration. Following are some examples based on field-flow fractionation technology.

Improved Multidetector AF4 Method for Particle Sizing and Concentration Measurements of Lipid-based Nanocarriers for RNA Delivery

Lipid-based nanoparticles for RNA delivery (LNP-RNA) are setting off a revolution in the field of nanomedicine. AF4 coupled to multiple online optical detectors (MD-AF4) is considered to be a very general and reliable method for the physical properties of nanocarriers and has been successfully used to measure the particle size, polydispersity, and physical stability of lipid-based systems, including liposomes and solid lipid nanoparticles. In addition to the analysis of particle size distribution, MD-AF4 can be used for extended characterization applications, including

  1. change in nanoparticle size distribution after protein binding;
  2. molecular weight distributions of polymeric NP components;
  3. release of free coatings/surfactants (e.g. PEG) from the surface of the particles;
  4. investigation of drug loading;
  5. and measurement of particle concentration.

As an additional objective, scientists apply MD-AF4 (in particular AF4-MALS) to measure the particle concentration and particle size distribution of lipid-based nanocarriers, supporting the results obtained by widely used techniques for particle concentration measurements such as nanoparticle tracking analysis (NTA). Overall, this work demonstrates the versatility of MD-AF4 in the characterization and quality control of lipid-based nanocarriers, widening its applicability to innovative systems such as LNP-RNA.

Number-based particle size distribution (particle/mL/nm) obtained by NTA and by AF4-MALS.Fig.1 Number-based particle size distribution (particle/mL/nm) obtained by NTA and by AF4-MALS. (Mildner, 2021)

Determination of Particle Size Distribution by AF4

Particle size distribution and stability are key indicators to evaluate the safety and effectiveness of medical nanoparticles (Med-NPs). In this work, AF4 is coupled to multiangle light scattering and dynamic light scattering detectors (AF4-MALS-DLS) as an alternative batch mode DLS to measure the physical properties of lipid-based nanoparticles. Interestingly, AF4-MALS-DLS analysis provides indirect information on particle shape (e.g., spherical vs elongated particles) by calculating the shape factor, which is the ratio of the geometric diameter (Dg) determined by MALS and the hydrodynamic diameter (Dh) measured by DLS. The calculated Dg to Dh ratio of all Lipidots formulations studied was in the 0.7-0.9 range, suggesting that they all possess a compact homogeneous core characterized by a spherical shape (theoretical compact sphere ratio=0.778). The AF4-MALS-DLS technique was able to successfully measure the PSD of the Lipidots with high resolution, being able to detect batch to batch variability induced by the manufacturing procedure, as well as to find out changes in the PSD induced by plasma proteins in complex biological media, avoiding the misinterpretation of the results which may result from batch mode DLS analysis. In conclusion, if a robust SOP is followed, AF4-MALS-DLS is a powerful method for the preclinical characterization of lipid-based nanoparticles.

Particle Size Distribution by AF4-MALS-DLS.Fig.2 Particle Size Distribution by AF4-MALS-DLS. (Caputo, 2019)

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  1. Mildner, R.; et al. Improved multidetector asymmetrical-flow field-flow fractionation method for particle sizing and concentration measurements of lipid-based nanocarriers for RNA delivery. European Journal of Pharmaceutics and Biopharmaceutics. 2021, 163: 252-265.
  2. Caputo, F.; et al. Measuring particle size distribution by asymmetric flow field flow fractionation: a powerful method for the preclinical characterization of lipid-based nanoparticles. Molecular pharmaceutics. 2019, 16(2): 756-767.
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