A Fast and Reliable Method Based on QCM-D Instrumentation for the Screening of Nanoparticle/Blood Protein Interactions

Abstract

The interactions that nanoparticles have with blood proteins are crucial for their fate in vivo. Such interactions result in the formation of the protein corona around the nanoparticles, and studying them aids in nanoparticle optimization. Quartz crystal microbalance with dissipation monitoring (QCM-D) can be used for this study. The present work proposes a QCM-D method to study the interactions on polymeric nanoparticles with three different human blood proteins (albumin, fibrinogen and γ-globulin) by monitoring the frequency shifts of sensors immobilizing the selected proteins. Bare PEGylated and surfactant-coated poly-(D,L-lactide-co-glycolide) nanoparticles are tested. The QCM-D data are validated with DLS and UV-Vis experiments in which changes in the size and optical density of nanoparticle/protein blends are monitored. We find that the bare nanoparticles have a high affinity towards fibrinogen and γ-globulin, with measured frequency shifts around -210 Hz and -50 Hz, respectively. PEGylation greatly reduces these interactions (frequency shifts around -5 Hz and -10 Hz for fibrinogen and γ-globulin, respectively), while the surfactant appears to increase them (around -240 Hz and -100 Hz and -30 Hz for albumin). The QCM-D data are confirmed by the increase in the nanoparticle size over time (up to 3300% in surfactant-coated nanoparticles), measured by DLS in protein-incubated samples, and by the trends of the optical densities, measured by UV-Vis. The results indicate that the proposed approach is valid for studying the interactions between nanoparticles and blood proteins, and the study paves the way for a more comprehensive analysis of the whole protein corona.

Keywords: QCM-D; blood proteins; hemocompatibility; nanoparticles; protein corona.

Figure 1

Schematization of QCM-D sensor functionalization (not in scale): in Step 1, the bare Au electrode is functionalized with a 12-MCA solution containing DTT as reducing agent, to provide the adlayer with carboxylic acid functionalities exposed over the surface; in Step 2, the carboxylic acid groups are activated via EDCl/NHS chemistry to provide N-hydroxy succinimide esters; in Step 3, the activated adlayer reacts with the selected blood protein to provide the probe layer.

Figure 2

Nanoparticle preparation: (a) chemical formulas of reagents (poly(D,L-lactide-co-glycolide carboxyl-terminated, PLGA-COOH, methoxy poly(ethylenglycol)-block-poly(D,L-lactide-co-glycolide, MPEG-b-PLGA, sodium cholate hydrate as surfactant) and corresponding schematization of the formed nanoparticles; (b) size and (c) surface ζ-potential, measured in samples containing freshly prepared nanoparticles.

Figure 3

QCM-D analysis: (a) traces of ΔF (black) and ΔD (red) registered in the experiments (only one plot is reported as a representative example. In this example, the probe was HPF, and the analyzed sample was PLGA-COOH + sodium cholate), the third overtone is reported (events: 1 pre-rinsing with water/ethanol, 2 injection of the adlayer solution, 3 rinsing with water/ethanol, 4 rinsing with water, 5 injection of the activation solution, 6 rinsing with water, 7 pre-rinsing with PBS, 8 injection of the probe solution, 9 rinsing with PBS, 10 rinsing with water, 11 pre-rinsing with PBS, 12 injection of the sample, 13 rinsing with PBS, 14 rinsing with water); (b) ΔF and (c) ΔD measured for the adlayer formation after rinsing (baseline water, n = 40); (d) ΔF and (e) ΔD measured for the probes after rinsing (baseline water, n = 14 for HSA, n = 12 for HPF and HBG); (f) areal masses of the probes, calculated with the Sauerbrey equation (Equation (1)) for all the overtones considered. In all the reported plots, red crosses are the outliers.

Figure 4

Sample detection and analysis of the transient sample/probe interactions: (a–c) ΔF shifts after rinsing, measured for samples PLGA-COOH, mPEG-b-PLGA, and PLGA-COOH + sodium cholate, respectively (n = 4 for each sample); (d–f) ΔD₃ vs. ΔF₃ measured during the detection, values are reported as the mean of 4 sensors, pre-rinsing and rinsing phases were excluded.

Figure 5

DLS analysis: mean size values measured vs. time for (a–c) PLGA-COOH; (d–f) mPEG-b-PLGA; and (g–I) PLGA-COOH + sodium cholate, respectively; (j–l) photon count rates.

Figure 6

UV-Vis analysis: optical densities measured once resuspended, and after 30 min and 60 min of incubation in the selected blood protein: (a) PLGA-COOH, (b) mPEG-b-PLGA, and (c) PLGA-COOH + sodium cholate.