Figure 1.
Strategies of PS NPs functionalization with Tf.
Cartoon representation of 100 nm polystyrene NPs modified with sulphonated groups (PSOSO3H, upper) and carboxylic groups (PSCOOH, down) coated with Tf by either physical adsorption (Tf@PSOSO3H and Tf@PSCOOH) or covalent coupling (Tf-PSCOOH): A, pristine NPs; B, Tf coated NPs; C, Tf coated NPs after incubation in human plasma. Both Tf@PSOSO3H and Tf-PSCOOH NPs are stable in human plasma, while Tf on Tf@PSCOOH NPs is easily replaced by environmental proteins.
Table 1.
DLS analysis of bare and Tf coated polystyrene nanoparticles in PBS (pH 7.4) at 25°C.
Figure 2.
Characterization of Tf coated NPs in PBS at room temperature.
Differential centrifugal sedimentation (DCS) results for: A, 100 nm PSOSO3H NPs and Tf@PSOSO3H NPs; B, 100 nm PSCOOH NPs and Tf@PSCOOH NPs and C, 100 nm PSCOOH NPs and Tf-PSCOOH. All size distributions show a shift towards larger apparent sizes in the presence of Tf coating. D, immuno detection of Tf coated NPs against monoclonal anti-Tf and anti-HAS.
Table 2.
DCS characteristics of Tf precoated NPs; hard corona (HC) and full corona (FC).
Figure 3.
DCS measurements of Tf coated NP-corona complexes before and after incubation in plasma.
A–C, apparent size comparison between Tf coated NPs dispersed in PBS, NPs incubated in plasma and isolated by spinning (so called hard corona) and NPs incubated in plasma “in situ” (so called full corona); D–F, the particle-corona complexes were isolated after either 5 min or 1 hour of incubation in the biological fluid. DCS experiment for the same NPs in PBS is reported for comparison in all graphs.
Figure 4.
Evaluation of Tf content in the NP coatings by SDS-PAGE and Western Blot.
Analysis of the protein coating in PBS and after incubation in 55% human plasma. A, SDS-PAGE of Tf@PSOSO3H NPs in PBS after 5 min and 1 hour of incubation in human plasma (after washing). Protein coronas of the pristine PSOSO3H NPs after 5 min and 1 hour incubation are reported as reference; B, western blot analysis of the gel reported in Figure A against polyclonal anti-Tf and densitometry of the bands; C, SDS-PAGE of Tf@PSCOOH and Tf-PSCOOH NPs in PBS and after 5 min and 1 hour of incubation in human plasma (after washing). Protein coronas of the pristine PSCOOH NPs after 5 min and 1 hour incubation are reported as reference; D, western analysis of the gel reported in panel C against polyclonal anti-Tf and densitometry of the bands.
Table 3.
Mass spectrometry data for human Tf amount on precoated PS nanoparticles before and after incubation in human plasma.
Figure 5.
Human plasma protein dot-blots of Tf coated NPs and their HC in 55% plasma.
1 µM (a) and 0.1 µM (b) solutions of human plasma proteins were spotted on the membrane to interact with functionalized NPs. Protein list: 1) HSA 2) Fibrinogen 3) Apo-E 4) Apo-AI 5) Plasminogen 6) Apo-B 7) Apo-AII 8) IgM 9) IgG and 10) Apo-C1.
Figure 6.
Tf@PSOSO3H and PSOSO3H NPs incubated on protein arrays.
Images of complete protein microarrays incubated with: A, 40 µg/ml PSOSO3H NPs in PBS (and sub-array 9); B, 40 µg/ml PSOSO3H in 10% human plasma (and miniature of sub-array 9); C, 40 µg/ml Tf@PSOSO3H NPs in PBS (and sub- array 26); D, 40 µg/ml Tf@PSOSO3H NPs in 10% human plasma (and miniature of sub-array 26).
Figure 7.
Human protein arrays positive hits comparison.
A, graph of total positive hits for bare and functionalized NPs in PBS and 10% human plasma solution; B, fluorescence intensity comparison for positive hits, between bare PSOSO3H NPs and Tf@PSOSO3H in PBS; C, fluorescence intensity comparison for positive hits, between PSOSO3H NPs and Tf@PSOSO3H in human plasma.
Table 4.
The fluorescence intensity for positive interaction hits for Tf@PSOSO3H in 10%plasma and PBS.