Deciphering the Emulsification Process to Create an Albumin-Perfluorocarbon-(o/w) Nanoemulsion with High Shelf Life and Bioresistivity

Abstract

This work aimed at the development of a stable albumin-perfluorocarbon (o/w) emulsion as an artificial oxygen carrier suitable for clinical application. So far, albumin-perfluorocarbon-(o/w) emulsions have been successfully applied in preclinical trials. Cross-linking a variety of different physical and chemical methods for the characterization of an albumin-perfluorocarbon (PFC)-(o/w) emulsion was necessary to gain a deep understanding of its specific emulsification processes during high-pressure homogenization. High-pressure homogenization is simple but incorporates complex physical reactions, with many factors influencing the formation of PFC droplets and their coating. This work describes and interprets the impact of albumin concentration, homogenization pressure, and repeated microfluidizer passages on PFC-droplet formation; its influence on storage stability; and the overcoming of obstacles in preparing stable nanoemulsions. The applied methods comprise dynamic light scattering, static light scattering, cryo- and non-cryo-scanning and transmission electron microscopies, nuclear magnetic resonance spectroscopy, light microscopy, amperometric oxygen measurements, and biochemical methods. The use of this wide range of methods provided a sufficiently comprehensive picture of this polydisperse emulsion. Optimization of PFC-droplet formation by means of temperature and pressure gradients results in an emulsion with improved storage stability (tested up to 5 months) that possibly qualifies for clinical applications. Adaptations in the manufacturing process strikingly changed the physical properties of the emulsion but did not affect its oxygen capacity.

Figure 1

Analysis of the droplet formation: (A) Increase in BSA concentration leads to accelerated droplet formation resulting in decreased droplet size. (B) ¹H NMR spectrum of the suspension. BSA gives rise to broad ¹H resonances between 10 and 0 ppm. Removing excess BSA from the suspension leads to the disappearance of all BSA-¹H signals. BSA bound to the emulsion droplets cannot be detected, presumably because of the much slower molecular tumbling due to the increased size of the droplets. (C) Increasing the emulsification pressure (EP) leads to initially smaller droplets that end up being the same size as droplets that form under lower EP. (D) Increasing the passage count (PC) from 1 to 15 led to initially smaller droplets that showed accelerated growth and ended up being bigger than those that formed during only one passage. (E) Increasing the PC led to a broader droplet size distribution (DSD) reflected by a higher polydispersity index (PDI) right after synthesis. (F) Interrelation between BSA consumption and the EP and PC during formation of the droplet. The average DSD (bars) determined by dynamic light scattering and the consumption of BSA (points) determined by bromocresol green staining and subsequent photometric determination are plotted as a function of EP and PC. With increasing EP and PC, the average droplet size decreased. The consumption of BSA increased with increasing PC and decreased with increasing EP. Mean values ± SD from n = 5; *p < 0.05, **p < 0.002, and ****p < 0.0001; one way analysis of variance (ANOVA) compared to the control followed by Dunnett′s post hoc test.

Figure 2

(A) Samples were stored in closed reaction vessels for 119 days at 4 °C. 1D ¹H NMR spectra were recorded on days 0, 7, 14, 43, 77, and 119. (B) Sample was stored in an unsealed NMR tube at 4 °C for 119 days. 1D ¹H NMR spectra were recorded on days 0, 7, 14, 43, 77, and 119.

Figure 3

(A) Intensity-mode DLS diagram of DSD showing a mean diameter of around 223 nm. (B) Interference microscopy image of an emulsion whose droplets are visible down to a diameter of 1 μm. The limit of detection for this microscopic method is about 300 nm; therefore, all visible droplets have diameters above the average diameter determined in DLS and SLS. (C) Volume-mode SLS diagram of the DSD showing a bimodal distribution. One population at around 150 nm and one population between 2 and 20 μm. (D) Size determination of albumin-PFC emulsion droplets using cSEM. Measurement of the shell thickness of BSA-PFC emulsion droplets after breakage during sublimation of PFC. (E)  cTEM image of the emulsion droplets emulsified with static temperature (4 °C) and a single passage at 20,000 PSI. (F)  cTEM image of emulsion droplets emulsified using a temperature and pressure gradient. (G) SLS diagram of repeated measurements presenting the DSD of two emulsion droplets emulsified using a temperature and pressure gradient on day 1 (white and light gray) and 5 months after emulsification (dark gray) in volume mode.

Figure 4

(A) Oxygen transfer: injection of 50 μL of preoxygenated PFC emulsion was able to increase the oxygen levels in 1 mL of blood by around 2.26 mL of O₂ (n = 6, t-test, p = 0,006). (B) Schematic visualization of the DOT. (C) TEM image of a THP-1-derived macrophage with incorporated emulsion droplets emulsified using a temperature and pressure gradient with HSA as a surfactant. (D) TEM image of incorporated HSA–emulsion droplets. After 4 h of incubation, the droplets remain in their primal shape and size.