In complex physiological media proteins form transient complexes with nanoparticles (NPs), mediated by competitive binding between proteins and NP surfaces, leading to the formation of a stable (hard) protein corona (HPC). Understanding the formation and the dynamics of this interaction is crucial for designing NP-based therapies, since HPC determines the biological identity of the NPs in vivo. The strong affinity between NPs surfaces and proteins can compensate the destabilization forces that colloidal NPs experience in high ionic strength media, stabilizing them. This interaction is immediate (soft -non stable –PC) and evolves with time (HPC). Nowadays, different studies regarding HPC composition show contradictory results. The complexity of serum composition, being NP size in the same range of proteins, and lack of reliable methods to determine composition of HPC, are behind these controversies. Several underestimated parameters regarding the response of NPs in physiological media (aggregation, dissolution) are critical determinants to be carefully addressed to better understand the formation of the HPC. In this context, it is necessary to develop simple but efficient and reliable protocols to study these processes. In this work, consequences of NP-PC formation providing a simple and reliable approach for determining both composition and physicochemical characterization of the HPC, and the implication for protein structures, is shown. In the first part, the hardening of the PC on 20 nm AuNPs, as a model case of metallic NP widely used in medicine, was monitored over time by UV-Vis spectroscopy, Dynamic Light Scattering and Z-Potential. Results of the process of HPC formation with only albumin or IgG were compared to results of HPC formation in serum. Time evolution of the NP-PC when conjugated with one protein can be understood as a fingerprint of the adsorption of that specific protein. Thus, the study of the PC evolution in serum provided information about the final composition of the HPC. Results showed similar pattern as when incubated when only albumin. Proteomic analysis confirmed the results. In addition, experiments mimicking the natural metabolic degradations of bioconjugates using etching agents (NaCN and HNO3), indicated that HPC exert protective effect on the NP core. Finally, limited proteolysis experiments indicated an altered metabolization of the protein inside the HPC, which can be related to a protein altered conformation in this adsorbed state. In the second part, HPC was studied on 50 nm SiO2 NPs, as a model case of metal oxide NP widely used in nanomedicine, by using either globular and intrinsically disordered proteins (IDPs), with the aim to investigate conformational changes induced by the interaction with NPs. IDPs exist in solution as conformational ensembles, whose features in the presence of NPs are still unknown. Three IDPs, acasein, Sic1 and asynuclein, were analyzed compared to lysozyme and transferrin (globular proteins model), describing conformational properties inside the HPC by circular dichroism and Fourier-transform infrared spectroscopy. Results indicated that IDPs maintain structural disorder inside HPC, experiencing minor, protein-specific, induced folding and stabilization against further conformational transitions. Oppositely, the analyzed globular proteins displayed the tendency to lose their ordered structure. Finally, the Transferrin-Tb complex, was also used in the HPC formation. The detection of the fluorescent properties of Tb upon HPC preparation is reported. By electrophoresis it was observed all the proteins forming the HPC and electron microscopy showed an HPC of a single layer of protein molecules. This latter part of work opens broad perspectives on the use of NP as agents that mimic macromolecular partners, allowing the comprehension of the effect of different factors affecting the interaction by rational design of NP surfaces.

In tamponi fisiologici, le proteine formano complessi transitori con nanoparticelle (NP), mediati da adsorbimento competitivo alla superficie di quest’ultime, fino alla formazione di una corona proteica stabile (HPC). Lo studio della dinamica d’interazione è fondamentale per ideare terapie basate su NP, in quanto l'HPC determina l'identità biologica delle NP in vivo. La forte affinità tra superfici di NP e proteine può compensare la destabilizzazione che le NP sperimentano in tamponi ad elevata forza ionica, stabilizzandole. Quest’interazione è immediata (soft -non stabile -PC) ma evolve nel tempo (HPC). Numerosi approcci volti a studiare la composizione dell'HPC hanno raffigurato uno scenario confuso, a causa delle dimensioni delle NP (comparabili con proteine) e della complessità composizionale del siero. Molti di questi studi non offrono un metodo affidabile per determinare la composizione dell'HPC e in alcuni casi sono contraddittori. La scarsa conoscenza della risposta dei nanomateriali in mezzi biologici è un punto chiave di queste controversie. Numerosi parametri, come l’aggregazione, sono sottostimati, ma risultano critici per comprendere la formazione dell'HPC. È necessario quindi sviluppare un protocollo semplice ma efficiente ed affidabile per studiare questi processi. In questo lavoro si è studiata la formazione di NP-PC con un approccio semplice e attendibile, al fine di determinare composizione e proprietà fisicochimiche dell'HPC oltre alle propriteà strutturali delle proteine adsorbite. Nella prima parte è studiato l’evoluzione nel tempo del PC su NP di oro 20 nm, come modello di NP metalliche utilizzato in medicina, monitorando evoluzione di proprietà fisicochimiche del PC con spettroscopia UV-Vis, Dynamic Light Scattering, Z-Potential. L’evoluzione dell’HPC proveniente dall'incubazione in siero è confrontata con quella risultante dall'incubazione con solo albumina o IgG, le proteine sieriche più abbondanti. L'evoluzione del PC quando coniugato con una proteina, è inteso come un'impronta digitale dell'adsorbimento di quella specifica proteina. Pertanto, questo confronto suggerisce la composizione finale dell'HPC. La dinamica del PC proveniente dal siero esibisce la dominanza fisicochimica dell'albumina, con poche differenze forse collegate alla presenza di composti minori nell’HPC. L'analisi proteomica conferma il risultato. L'HPC mantiene le sue caratteristiche nel tempo ed esercita un effetto protettivo sul nucleo della NP se introdotte in condizioni di attacco chimico (NaCN e HNO3) che mimano la degradazione metabolica del PC. La proteolisi limitata dell’HPC indica un’alterata metabolizzazione proteica all'interno dell'HCC, probabilmente dovuta ad una sua variazione strutturale. Nella seconda parte, HPC è studiato su NP di SiO2 di 50 nm, come modello di NP di ossido utilizzato in nanomedicina, utilizzando sia proteine globulari che intrinsecamente disordinate (IDP), con lo scopo di indagare cambiamenti conformazionali indotti dall'interazione con NP. Le IDP esistono in soluzione in un insieme conformazionale, le cui caratteristiche in presenza di NP sono sconosciute. Tre IDP, a-caseina, Sic1 e a-sinucleina sono state analizzate rispetto a lisozima e transferrina (proteine globulari modello). Le struttura nell’HPC è studiata mediante dicroismo circolare e spettroscopia infrarossa a trasformata di Fourier. I risultati indicano che le IDP mantengono il disordine strutturale all'interno dell'HPC, sperimentando minori transizioni conformazionali ma essendo stabilizzate contro variazioni dell’intorno. Le globulari, invece, tendono a perdere la loro struttura ordinata. Le proteine nell'HPC sono visualizzate mediante elettroforesi mentre microscopia elettronica mostra un HPC formato da un singolo strato di molecole proteiche. Quest'ultima parte apre ampie prospettive sull'uso di NP come agenti che imitano partner molecolari.

(2018). Dynamics of nanoparticle-protein corona: formation, evolution and insight on protein structure. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).

Dynamics of nanoparticle-protein corona: formation, evolution and insight on protein structure

VITALI, MICHELE
2018

Abstract

In complex physiological media proteins form transient complexes with nanoparticles (NPs), mediated by competitive binding between proteins and NP surfaces, leading to the formation of a stable (hard) protein corona (HPC). Understanding the formation and the dynamics of this interaction is crucial for designing NP-based therapies, since HPC determines the biological identity of the NPs in vivo. The strong affinity between NPs surfaces and proteins can compensate the destabilization forces that colloidal NPs experience in high ionic strength media, stabilizing them. This interaction is immediate (soft -non stable –PC) and evolves with time (HPC). Nowadays, different studies regarding HPC composition show contradictory results. The complexity of serum composition, being NP size in the same range of proteins, and lack of reliable methods to determine composition of HPC, are behind these controversies. Several underestimated parameters regarding the response of NPs in physiological media (aggregation, dissolution) are critical determinants to be carefully addressed to better understand the formation of the HPC. In this context, it is necessary to develop simple but efficient and reliable protocols to study these processes. In this work, consequences of NP-PC formation providing a simple and reliable approach for determining both composition and physicochemical characterization of the HPC, and the implication for protein structures, is shown. In the first part, the hardening of the PC on 20 nm AuNPs, as a model case of metallic NP widely used in medicine, was monitored over time by UV-Vis spectroscopy, Dynamic Light Scattering and Z-Potential. Results of the process of HPC formation with only albumin or IgG were compared to results of HPC formation in serum. Time evolution of the NP-PC when conjugated with one protein can be understood as a fingerprint of the adsorption of that specific protein. Thus, the study of the PC evolution in serum provided information about the final composition of the HPC. Results showed similar pattern as when incubated when only albumin. Proteomic analysis confirmed the results. In addition, experiments mimicking the natural metabolic degradations of bioconjugates using etching agents (NaCN and HNO3), indicated that HPC exert protective effect on the NP core. Finally, limited proteolysis experiments indicated an altered metabolization of the protein inside the HPC, which can be related to a protein altered conformation in this adsorbed state. In the second part, HPC was studied on 50 nm SiO2 NPs, as a model case of metal oxide NP widely used in nanomedicine, by using either globular and intrinsically disordered proteins (IDPs), with the aim to investigate conformational changes induced by the interaction with NPs. IDPs exist in solution as conformational ensembles, whose features in the presence of NPs are still unknown. Three IDPs, acasein, Sic1 and asynuclein, were analyzed compared to lysozyme and transferrin (globular proteins model), describing conformational properties inside the HPC by circular dichroism and Fourier-transform infrared spectroscopy. Results indicated that IDPs maintain structural disorder inside HPC, experiencing minor, protein-specific, induced folding and stabilization against further conformational transitions. Oppositely, the analyzed globular proteins displayed the tendency to lose their ordered structure. Finally, the Transferrin-Tb complex, was also used in the HPC formation. The detection of the fluorescent properties of Tb upon HPC preparation is reported. By electrophoresis it was observed all the proteins forming the HPC and electron microscopy showed an HPC of a single layer of protein molecules. This latter part of work opens broad perspectives on the use of NP as agents that mimic macromolecular partners, allowing the comprehension of the effect of different factors affecting the interaction by rational design of NP surfaces.
GRANDORI, RITA
PUNTES, VICTOR FRANCO
nanoparticles,; protein-corona,; time-evolution,; structural-disorder,; mimic-effect
nanoparticles,; protein-corona,; time-evolution,; structural-disorder,; mimic-effect
BIO/10 - BIOCHIMICA
English
15-mar-2018
SCIENZA E NANOTECNOLOGIA DEI MATERIALI - 79R
30
2016/2017
open
(2018). Dynamics of nanoparticle-protein corona: formation, evolution and insight on protein structure. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/199091
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