Influence of bound dodecanoic acid on the reconstitution of albumin nanoparticles from a lyophilized state

Abstract

The development of reference standards for nanoparticle sizing allows for cross laboratory studies and effective transfer of particle sizing methodology. To facilitate this, these reference standards must be stable upon long-term storage. Here, we examine factors that influence the properties of cross-linked albumin nanoparticles, fabricated with an ethanol desolvation method, when reconstituted from a lyophilized state. We demonstrate, with nanoparticle tracking analysis, no significant changes in mean particle diameter upon reconstitution of albumin nanoparticles fabricated with bovine serum albumin loaded with dodecanoic acid, when compared to nanoparticles fabricated with a fatty acid-free BSA. We attribute this stability to the modulation of nanoparticle charge-charge interactions at dodecanoic acid specific binding locations. Furthermore, we demonstrate this in a lyophilized state over six months when stored at - 80 °C. We also show that the reconstitution process is readily transferable between technicians and laboratories and further confirm our finding with dynamic light scattering analysis.

Figure 1

(A) Distribution profiles of BSA-DF (red) and BSA-DF-C12 (black) nanoparticle before lyophilization. (B) Distribution profiles after reconstitution from a lyophilized state for BSA-DF (black), BSA-DF 33 (pink), BSA-DF 50 (blue), BSA-DF 100 (cyan) and BSA-DF 200 (red). (C) Distribution profiles after reconstitution from a lyophilized state for BSA-DF-C12 (black), BSA-DF-C12 10 (pink), BSA-DF-C12 33 (blue), BSA-DF-C12 50 (cyan) and BSA-DF-C12 100 (red). Plots represent the mean from three measurements with nanoparticle tracking analysis.

Figure 2

(A) Plots of mean diameter (grey bars) and Span (black circles) pre freeze-drying (BSA-DF) and after reconstitution at various particle concentrations for BSA nanoparticles fabricated with defatted protein. (B) Plot of particle recovery for freeze-dried BSA-DF nanoparticles at different concentrations upon reconstitution. Data represents mean values from three measurements and error bars represent the standard deviation.

Figure 3

(A) Plots of mean diameter (grey bars) and Span (black circles) pre freeze-drying (BSA-DF-C12) and after reconstitution at various particle concentrations for BSA nanoparticles fabricated with dodecanoic acid loaded protein. (B) Plot of particle recovery for freeze-dried BSA-DF-C12 nanoparticles at different concentrations upon reconstitution. Data represents mean values from three measurements and error bars represent the standard deviation.

Figure 4

Atomic force microscopy images of BSA-DF-C12 lyophilized sample spin-coated and measured in ambient.