Real-time monitoring of protein–liposome interaction kinetics using absorption, polarized intrinsic emission, and scattering (APIES): insights into protein corona formation

Liposome stability is strongly influenced by rapid protein adsorption once they enter biological environments. This dynamic "protein corona" alters membrane properties, permeability, and circulation behaviour. The early stages of protein-liposome interactions and the specific processes involved are not often studied. Addressing this gap, we report the first application of simultaneous absorption, polarized intrinsic emission (280 nm excitation), and scattering (APIES) spectroscopy with rapid mixing to monitor in real time, with second-level resolution the interaction of Human serum albumin (HSA) with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes (D_h ∼150-160 nm) in ammonium bicarbonate buffer of varying ionic strength (25 to 150 mM). Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) were used to measure changes in particle size distribution (PSD). APIES clearly showed a three-phase interaction process: first rapid (0-200 seconds) protein adsorption without penetration and osmotic pressure induced size decrease (∼5 to 10%); second, a penetration phase (up to ∼30 minutes) where HSA enters the lipid bilayer, and third, an annealing phase (up to 3 hours) where HSA-DMPC particle size gradually increased (by ∼2 to 5%). APIES characterized the first phase by constant absorbance (i.e. no protein loss), minimal spectral shifts, but small reductions in emission intensity and ratio of fluorescence band area to Rayleigh-Mie scatter band area (A_FI/Ry). Phase two displayed constant absorbance, minimal changes in A_FI/Ry, but relatively large blue shifts (indicative of tryptophan environment changes). The final phase displayed constant absorbance, gradual increase in A_FI/Ry (which correlates linearly with particle size) and fluorescence intensity with minimal emission maxima changes. APIES also clearly resolved differences due to ionic strength (electrostatic screening effects) by A_FI/Ry, (decreased by ∼20% at 25 mM compared to <5% at 100-150 mM after 30 minutes) hypsochromic spectral shifts (5.9 ± 1.2 nm at 25, 3.0 ± 0.8 nm at 150 mM after 3 hours). DLS confirmed that HSA-DMPC size decreased significantly within 200 seconds after mixing, and the gradual size increase afterwards. NTA confirmed the effect of ionic strength on HSA-liposome complex size and PSD. In conclusion, APIES is a powerful, yet simple tool for monitoring protein corona formation in real time (~1 s resolution), providing more detailed mechanistic insights beyond PSD changes.