Nano Differential Scanning Fluorimetry as a Rapid Stability Assessment Tool in the Nanoformulation of Proteins

Abstract

The development and production of innovative protein-based therapeutics is a complex and challenging avenue. External conditions such as buffers, solvents, pH, salts, polymers, surfactants, and nanoparticles may affect the stability and integrity of proteins during formulation. In this study, poly (ethylene imine) (PEI) functionalized mesoporous silica nanoparticles (MSNs) were used as a carrier for the model protein bovine serum albumin (BSA). To protect the protein inside MSNs after loading, polymeric encapsulation with poly (sodium 4-styrenesulfonate) (NaPSS) was used to seal the pores. Nano differential scanning fluorimetry (NanoDSF) was used to assess protein thermal stability during the formulation process. The MSN-PEI carrier matrix or conditions used did not destabilize the protein during loading, but the coating polymer NaPSS was incompatible with the NanoDSF technique due to autofluorescence. Thus, another pH-responsive polymer, spermine-modified acetylated dextran (SpAcDEX), was applied as a second coating after NaPSS. It possessed low autofluorescence and was successfully evaluated with the NanoDSF method. Circular dichroism (CD) spectroscopy was used to determine protein integrity in the case of interfering polymers such as NaPSS. Despite this limitation, NanoDSF was found to be a feasible and rapid tool to monitor protein stability during all steps needed to create a viable nanocarrier system for protein delivery.

Keywords: bovine serum albumin; mesoporous silica nanoparticles; microfluidics; nano differential scanning fluorimetry; protein encapsulation; protein stability.

Figure 1

Schematic representation of the synthesis of nanoparticles and their polymer encapsulation by microfluidics.

Figure 2

TEM images of the synthesized and microfluidic-coated MSNs: (A) Plain MSNs, scale bar: 200 nm; (B) Loaded MSN-PEI–BSA, scale bar: 200 nm; (C) MSN-PEI–BSA–NaPSS, scale bar: 100 nm; (D) MSN-PEI–BSA–NaPSS–SpAcDEX, scale bar: 100 nm.

Figure 3

Thermal unfolding curves of BSA in different conditions, measured using NanoDSF. The F350/F330 ratio and its first derivative is shown for each sample. Each curve is averaged from three experiments. Melting temperatures (T_m) correspond to the minimum of the first derivative. (A) The effect of PEI-functionalized MSNs on the stability of BSA; (B) the effect of the SpAcDEX polymer; (C) the effect of increasing concentrations of ethanol; (D) the effect of different buffers and pH; and (E) the effect of surfactants and salts. The curves in (F) resulted from the autofluorescence of the NaPSS polymer, and do not represent its effect on BSA.

Figure 4

CD spectrum of BSA (black line) and BSA in the presence of NaPSS at different concentrations in DI water (pH 7.0). Optical path length 1.0 mm. The plotted curves are averaged from the three experiments ± StD.