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The properties of nanoparticles (NPs) can change upon contact with serum components, occluding the NP surface by forming a biomolecular corona. It is believed that targeted NPs can lose their functionality due to this biological coating, thus losing specificity and selectivity toward target cells and leading to poor therapeutic efficiency. A better understanding of how the biomolecular corona affects NP ligand functionality is needed to maintain NP targeting capabilities. However, techniques that can quantify the functionality of NPs at a single-particle level in a complex medium are limited and often laborious in sample preparation, measurement, and analysis. In this work, the influence of serum exposure on the functionality of antibody-functionalized NPs was quantified using a straightforward total internal reflection fluorescence (TIRF) microscopy method and evaluated in cell uptake studies. The single-particle resolution of TIRF reveals the interparticle functionality heterogeneity and the substantial differences between NPs conjugated with covalent and noncovalent methods. Notably, only NPs covalently conjugated with a relatively high amount of antibodies maintain their functionality to a certain extent and still showed cell specificity and selectivity toward high receptor density cells after incubation in full serum. The presented study emphasizes the importance of single-particle functional characterization of NPs in complex media, contributing to the understanding and design of targeted NPs that retain their cell specificity and selectivity in biologically relevant conditions.

Methodological constraints have limited our ability to study protein corona formation, slowing nanomedicine development and their successful translation into the clinic. We determined hard and soft corona structural properties along with the corresponding proteomic compositions on liposomes in a label-free workflow: surface plasmon resonance and a custom biosensor for in situ structure determination on liposomes and corona separation, and proteomics using sensitive nanoliquid chromatography tandem mass spectrometry with open-source bioinformatics platforms. Undiluted human plasma under dynamic flow conditions was used for in vivo relevance. Proof-of-concept is presented with a regular liposome formulation and two light-triggered indocyanine green (ICG) liposome formulations in preclinical development. We observed formulation-dependent differences in corona structure (thickness, protein-to-lipid ratio, and surface mass density) and protein enrichment. Liposomal lipids induced the enrichment of stealth-mediating apolipoproteins in the hard coronas regardless of pegylation, and their preferential enrichment in the soft corona of the pegylated liposome formulation with ICG was observed. This suggests that the soft corona of loosely interacting proteins contributes to the stealth properties as a component of the biological identity modulated by nanomaterial surface properties. The workflow addresses significant methodological gaps in biocorona research by providing truly complementary hard and soft corona compositions with corresponding in situ structural parameters for the first time. It has been designed into a convenient and easily reproducible single-experiment format suited for preclinical development of lipid nanomedicines.

Bio-nano interface investigation models are mainly based on the type of proteins present on corona, bio-nano interaction responses and the evaluation of final outcomes. Due to the extensive diversity in correlative models for investigation of nanoparticles biological responses, a comprehensive model considering different aspects of bio-nano interface from nanoparticles properties to protein corona fingerprints appeared to be essential and cannot be ignored. In order to minimize divergence in studies in the era of bio-nano interface and protein corona with following therapeutic implications, a useful investigation model on the basis of RADAR concept is suggested. The contents of RADAR concept consist of five modules: 1- Reshape of our strategy for synthesis of nanoparticles (NPs), 2- Application of NPs selected based on human fluid, 3- Delivery strategy of NPs selected based on target tissue, 4- Analysis of proteins present on corona using correct procedures and 5- Risk assessment and risk reduction upon the collection and analysis of results to increase drug delivery efficiency and drug efficacy. RADAR grouping strategy for revisiting protein corona phenomenon as a key of success will be discussed with respect to the current state of knowledge.

Due to late diagnosis, high incidence of metastasis, and poor survival rate, pancreatic cancer is one of the most leading cause of cancer-related death. Although manifold recent efforts have been done to achieve an early diagnosis of pancreatic cancer, CA-19.9 is currently the unique biomarker that is adopted for the detection, despite its limits in terms of sensitivity and specificity. To identify potential protein biomarkers for pancreatic ductal adenocarcinoma (PDAC), we used three model liposomes as nanoplatforms that accumulate proteins from human plasma and studied the composition of this biomolecular layer, which is known as protein corona. Indeed, plasma proteins adsorb on nanoparticle surface according to their abundance and affinity to the employed nanomaterial, thus even small differences between healthy and PDAC protein expression levels can be, in principle, detected. By mass spectrometry experiments, we quantified such differences and identified possible biomarkers for PDAC. Some of them are already known to exhibit different expressions in PDAC proteomes, whereas the role of other relevant proteins is still not clear. Therefore, we predict that the employment of nanomaterials and their protein corona may represent a useful tool to amplify the detection sensitivity of cancer biomarkers, which may be used for the early diagnosis of PDAC, with clinical implication for the subsequent therapy in the context of personalized medicine.

Nanoparticles (NPs) have emerged as an effective means to deliver anticancer drugs into the brain. Among various forms of NPs, liposomal temozolomide (TMZ) is the drug-of-choice for the treatment and management of brain tumours, but its therapeutic benefit is suboptimal. Although many possible reasons may account for the compromised therapeutic efficacy, the inefficient tumour penetration of liposomal TMZ can be a vital obstacle. Recently, the protein corona, i.e., the layer of plasma proteins that surround NPs after exposure to human plasma, has emerged as an endogenous trigger that mostly controls their anticancer efficacy. Exposition of particular biomolecules from the corona referred to as protein corona fingerprints (PCFs) may facilitate interactions with specific receptors of target cells, thus, promoting efficient internalization. In this work, we have synthesized a set of four TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical−chemical properties. We have demonstrated that precoating liposomal TMZ with a protein corona made of human plasma proteins can increase drug penetration in a 3D brain cancer model derived from U87 human glioblastoma multiforme cell line leading to marked inhibition of tumour growth. On the other side, by fine-tuning corona composition we have also provided experimental evidence of a non-unique effect of the corona on the tumour growth for all the complexes investigated, thus, clarifying that certain PCFs (i.e., APO-B and APO-E) enable favoured interactions with specific receptors of brain cancer cells. Reported results open new perspectives into the development of corona-coated liposomal drugs with enhanced tumour penetration and antitumour efficacy.

Nanoparticles are promising tools for nanomedicine in a wide array of therapeutic and diagnostic applications. Yet, despite the advances in the biomedical applications of nanomaterials, relatively few nanomedicines made it to the clinics. The formation of the biomolecular corona on the surface of nanoparticles has been known as one of the challenges toward successful targeting of nanomedicines. This adsorbed protein layer can mask targeting moieties and creates a new biological identity that critically affects the subsequent biological interactions of nanomedicines with cells. Extensive studies have been directed toward understanding the characteristics of this layer of biomolecules and its implications for nanomedicine outcomes at cell and organism levels, yet several aspects are still poorly understood. One aspect that still requires further insights is how the biomolecular corona interacts with and is "read" by the cellular machinery. Within this context, this review is focused on the current understanding of the interactions of the biomolecular corona with cell receptors. First, we address the importance and the role of receptors in the uptake of nanoparticles. Second, we discuss the recent advances and techniques in characterizing and identifying biomolecular corona-receptor interactions. Additionally, we present how we can exploit the knowledge of corona-cell receptor interactions to discover novel receptors for targeting of nanocarriers. Finally, we conclude this review with an outlook on possible future perspectives in the field. A better understanding of the first interactions of nanomaterials with cells, and -in particular -the receptors interacting with the biomolecular corona and involved in nanoparticle uptake, will help for the successful design of nanomedicines for targeted delivery.

Nanoparticles are promising tools for nanomedicine in a wide array of therapeutic and diagnostic applications. Yet, despite the advances in the biomedical applications of nanomaterials, relatively few nanomedicines made it to the clinics. The formation of the biomolecular corona on the surface of nanoparticles has been known as one of the challenges toward successful targeting of nanomedicines. This adsorbed protein layer can mask targeting moieties and creates a new biological identity that critically affects the subsequent biological interactions of nanomedicines with cells. Extensive studies have been directed toward understanding the characteristics of this layer of biomolecules and its implications for nanomedicine outcomes at cell and organism levels, yet several aspects are still poorly understood. One aspect that still requires further insights is how the biomolecular corona interacts with and is “read” by the cellular machinery. Within this context, this review is focused on the current understanding of the interactions of the biomolecular corona with cell receptors. First, we address the importance and the role of receptors in the uptake of nanoparticles. Second, we discuss the recent advances and techniques in characterizing and identifying biomolecular corona-receptor interactions. Additionally, we present how we can exploit the knowledge of corona-cell receptor interactions to discover novel receptors for targeting of nanocarriers. Finally, we conclude this review with an outlook on possible future perspectives in the field. A better understanding of the first interactions of nanomaterials with cells, and -in particular -the receptors interacting with the biomolecular corona and involved in nanoparticle uptake, will help for the successful design of nanomedicines for targeted delivery.

Namen magistrske naloge je preveriti, kakšen vpliv ima biomolekularna korona izbranih nanomaterialov na vzajemno delovanje med nanomateriali in celicami. Testirali smo vpliv nanodelcev (ND) s predhodno ustvarjeno korono iz govejega serumskega albumina - BSA (predhodno oblečeni ND) ter tistih brez predhodno ustvarjene korone (predhodno neoblečeni ND) na človeških nevroblastomskih celicah SH-SY5Y. Testiranje smo izvajali s sedmimi različnimi nanodelci – tremi nanodelci TiO2 z različnimi lastnostmi ter nanodelci SiO2, Ag, CuO in ZnO. Za preverjanje vpliva korone smo opravili tri teste citotoksičnosti – test z barvilom resazurin, test privzema barvila nevtralno rdeče in test z barvilom Coomassie Brilliant Blue. Preverili smo tudi, koliko proteinov se je vezalo v korono posameznih delcev s testom BCA za določanje količine prisotnih proteinov. Predhodno oblečeni ZnO in CuO nanodelci so bili bolj citotoksični, kot tisti brez predhodno ustvarjene korone, ali pa je bila citotoksičnost obeh tretmajev podobna – pri nanodelcih Ag, ali pa citotoksičnega učinka nismo opazili - TiO2 ter SiO2. Vpliv prehodno ustvarjene korone na nanodelcih je bil bolj izrazit pri nanodelcih CuO in ZnO, od katerih se odtapljajo ioni. Predvidevamo, da so razlike v učinku predhodno oblečenih in neoblečenih ND ZnO in CuO, posledica olajšane endocitoze predhodno oblečenih ND, vendar tega nismo preverjali. The purpose of this master's thesis was to test the effect that the biomolecular corona of chosen nanomaterials has on the interaction between them and the cells. We tested two groups of nanoparticles. For the first group, we incubated pristine nanoparticles in bovine serum albumin - BSA before testing, so a corona formed. For the second group we used nanoparticles of the same material without a preformed corona. We tested the effect of each group of nanoparticles on human neuroblastoma cells SH-SY5Y and tested seven different types of nanoparticles – SiO2, Ag, CuO, ZnO, and three different TiO2 nanoparticle samples. To asses the effect of the preformed protein corona versus no preformed corona on SH-SY5Y cells we used several cytotoxicity tests – resazurin test, neutral red uptake test (NR) and coomassie brilliant blue test (CBB). We also evaluated the amount of proteins bound to nanoparticles with a preformed corona using a BCA Protein Assay Test. We found that the ZnO and CuO nanoparticles with a preformed corona had a greater cytotoxic effect on cells, than those without a preformed corona. The cytotoxic effect of nanoparticles with and without the corona was simmilar in the case of Ag nanoparticles, and for the TiO2 ter SiO2 we did not notice significant cytotoxic effects. The effect of a preformed corona was more noticable in CuO and ZnO nanoparticles, which dissolute into ions. We presume that the differences in the effect of nanoparticles with and without a preformed corona are due to increased endocytosis of nanoparticles with a preformed corona, but this was not tested.

Biomolecules adsorbed on nanoparticles are known to confer a biological identity to nanoparticles, mediating the interactions with cells and biological barriers. However, how these molecules are presented on the particle surface in biological milieu remains unclear. The central aim of this study is to identify key protein recognition motifs and link them to specific cell-receptor interactions. Here, we employed an immuno-mapping technique to quantify epitope presentations of two major proteins in the serum corona, low-density lipoprotein and immunoglobulin G. Combining with a purpose-built receptor expression system, we show that both proteins present functional motifs to allow simultaneous recognition by low-density lipoprotein receptor and Fc-gamma receptor I of the corona. Our results suggest that the “labeling” of nanoparticles by biomolecular adsorption processes allows for multiple pathways in biological processes in which they may be “mistaken” for endogenous objects, such as lipoproteins, and exogenous ones, such as viral infections. European Commission - Seventh Framework Programme (FP7) Science Foundation Ireland Saudi Arabia Scholarship Program

Nanosized objects, such as nanoparticles and other drug carriers used in nanomedicine, once in contact with biological environments are modified by adsorption of biomolecules on their surface. The presence of this corona strongly affects the following interactions at cell and organism levels. It has been shown that corona proteins can be recognized by cell receptors. However, it is not known whether the composition of this acquired layer can also affect the mechanisms nanoparticles use to enter cells. This is of particular importance when considering that the same nanoparticles can form different coronas for instance in vitro when exposed to cells in different serum amounts or in vivo depending on the exposure or administration route. Thus, in this work, different coronas were formed on 50 nm silica by exposing them to different serum concentrations. The uptake efficiency in HeLa cells was compared, and the uptake mechanisms were characterized using transport inhibitors and RNA interference. The results showed that the nanoparticles were internalized by cells via different mechanisms when different coronas were formed, and only for one corona condition was uptake mediated by the LDL receptor. This suggested that coronas of different composition can be recognized differently by cell receptors, and this in turn leads to internalization via different mechanisms. Similar studies were performed using other cells, including A549 cells and primary HUVEC, and different nanoparticles, namely 100 nm liposomes and 200 nm silica. Overall, the results confirmed that the corona composition can affect the mechanisms of nanoparticle uptake by cells. This work was funded by the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme under grant agreement no. 637614 (NanoPaths). A.S. kindly acknowledges the University of Groningen for additional funding (Rosalind Franklin Fellowship). S.D. was supported by a postdoctoral fellowship granted by the Research Foundation Flanders (FWO) and the Flemish Institute for Technological Research (VITO) under grant agreement 12S6517N. Financial support from the China Scholarship Council to, K.Y. is also acknowledged. Mass spectrometry analysis was performed in the Interfaculty Mass Spectrometry Center of the University of Groningen (RUG) and University Medical Center Groningen (UMCG). The authors would like to thank H. Permentier and C. M. Jeronimus-Stratingh for technical help and suggestions for sample preparation for mass spectrometry.

This trends article describes the analytical approaches for the in-depth characterization of the protein corona on liposome nanoparticles. In particular, examples since 2014 are summarized according to the analytical approach. Traditional protein corona characterizations from in vitro static experiments are provided along with the newly introduced experimental setups for characterization of the protein corona by in vitro dynamic and in vivo studies. Additionally, a special attention is also devoted to the need for introduction of new experimental workflows for characterization of a much wider array of biomolecules. In the most recent years, an extension of the protein corona concept to the biomolecular corona was introduced, and the analytical targets are no longer restricted to proteins, but to other important biomolecules as well, as they can potentially affect the biodistribution and interaction of nanoparticles with the biological systems. The few recent examples in this field are discussed for the characterization of metabolites and lipids in the biomolecular corona with examples, also extending the discussion from liposome to other types of nanoparticles. A final discussion is provided on the potential key role of the most recent omics approaches in the study of the nano-bio interface, with an overview on top-down proteomics, which allows a better elucidation of proteoforms, and on lipidomics and metabolomics, which allow a comprehensive untargeted characterization of lipids and metabolites, respectively. Graphical abstract.

In physiological environments (e.g. the blood), nanoparticles (NPs) are surrounded by a layer of biomolecules referred to as a ‘protein corona’ (PC). The most tightly NP-bound proteins form the so-called hard corona (HC), the key bio-entity that determines the NP's biological identity and physiological response. To date, NP-HC has been almost exclusively characterized in vitro, while NP–protein interactions under realistic in vivo conditions remain largely unexplored. In this study, we thoroughly characterized the in vivo HC of a NP formulation that forms around lipid nanoparticles with a lipid composition equal to that of clinically used liposomal amphotericin B (AmBisome®) after the recovery of the NPs from the blood circulation of FVB/N mice 10 minutes post intravenous administration. In vitro HC formed by 10 minutes incubation of NPs in FVB/N mouse plasma was used for comparison. Here we show that the biological identity (i.e. size, zeta-potential and aggregation state) of NPs in vivo is significantly different from that acquired in vitro. Furthermore, the variety of protein species in the in vivo HC was considerably larger. The present work has demonstrated that characterization of the in vivo HC is essential to provide an accurate molecular description of the biological identity of NPs in physiological environments.

It has been well established that the early stages of nanoparticle cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions, nanoparticles are typically internalized into the cell and trafficked along defined pathways such as, in many cases, the endolysosomal pathway. Indeed, if the original corona is partially retained on the nano particle surface, the biomolecules in this layer may play an important role in determining subsequent cellular processing. In this work, using a combination of organelle separation and fluorescence labeling of the initial extracellular corona, we clarify its intracellular evolution as nanoparticles travel within the cell. We show that specific proteins present in the original protein corona are retained on the nanoparticles until they accumulate in lysosomes, and, once there, they are degraded. We also report on how different bare surfaces (amino and carboxyl modified) affect the details of this evolution. One overarching discovery is that the same serum proteins can exhibit different intracellular processing when carried inside cells by nanoparticles, as components of their corona, compared to what is observed when they are transported freely from the extracellular medium.

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (O≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.