Nanoparticles (NPs) are one of the most important tools in the emerging area of nanomedicine. The behavior of NPs in relevant biological environments, as preclinical setting, may be quite complex due to both their interactions with biological fluids and the formation of a protein layer, called protein corona (PC). PC remarkably affects the physicochemical properties of NPs (size, shape, surface chemistry, aggregation state, etc.) and consequently their biological fate including their pharmacokinetics, biodistribution, toxicity and therapeutic efficacy. Strong efforts were applied to correlate PC composition and observed effect after in vitro/in vivo experiments, but unfortunately poor reproducibility of data is often assessed. Biological, chemical and physical properties of NPs along with proteins composition of PC and features of pathological environments hardly complicate the study of interactions between PC-NP complexes and their interaction with target cells. In this contest, in order to quantitatively and qualitatively evaluate PC adsorbed onto NPs, the optimization of purification/separation procedures and rigorous and standardized analytical methods become a priority request to further design tailored nanomaterial able to interact with proteins and cells in a tunable manner.
Keywords: Nanocarriers, Molecular size, Nanoparticles, Pharmacokinetics, NP complexes
Nanomedicine is one of the most active research areas of nanotechnology involving the application of nanocarriers for the medical prevention, diagnosis, and treatment of diseases . Polymeric or inorganic nanoparticles (NPs) have the capacity to incorporate active substances of various characteristics and protect them from the inhospitable biological environment . Due to their nanometrical size, NPs show ability to cross tissue barriers and the cell membrane, thus allowing interaction with the smaller components such as cellular proteins and other macromolecules . The production of PEG-decorated NPs could reduce the reticuloendothelial system (RES) uptake, accumulation in liver, spleen or bone marrow, increasing circulation time and limit non-specific target uptake ultimately leading to a decrease in toxicity . Further functionalization with targeting ligands, which possess the inherent ability to facilitate selective binding to cell types, can confer “smart” properties to NPs .
Generally, to rationally design an efficient nanoparticle-based therapeutic tool able to selectively transfer drugs to the target site, thus minimizing side effects and increasing therapeutic compliance, the combination of the nanocarriers formulative aspects along with a fundamental understanding of the molecular mechanism involved in regulating nanocarrier-biological interactions is highly required. In fact, immediately after NPs come into contact with protein-containing media (such as biological fluids), a layer of proteins, called the protein corona (PC), is formed on the particle surface. This PC could remarkably alter the original Np molecular identity affecting their clearance by RES, cellular uptake, biodistribution and also toxicity. Furthermore, if the NPs are surface-functionalized with selective ligands, the absorption of proteins could mask the targeting ability, inhibiting their biological effects. Thus, it is more than evident that the PC plays a key role in the interaction of particles with cells after systemic administration .
The PC has recently been the subject of extensive studies aiming to investigate it as complex and multiple layer entity characterized by proteins that exchange dynamically between the surface and the surrounding environment (soft corona-SC) and proteins more firmly adsorbed on the surface of the NPs (hard corona-HC) [7,8]. An unsolved issue remains in identifying both the stability and the role of these protein layers in biodistribution and the efficacy effects in transferring drugs to target tissues and cells. The highlights summary listed in Table 1 on the evidence of biological effects in function of the PC’s on the NPs (also including liposomes and nanotubes) strongly confirms this difficulty. These data obtained generally by in vitro experiments, simulating in vivo biological conditions, are affected by both the nanocarrier features (material, size, surface, charge, shape) and environment (composition, exposure time, pH, temperature, shear stress). Unfortunately, considering all these variables, the data obtained by different research groups seem to be poorly reproducible, frequently not useful or premonitory for completely clarifying the role of the PC and to better tailor design and produce really efficacious drug targeting carriers.
As evidence, conflicting reports on cytotoxicity and biological fates were reported even when similar NPs were tested [9-12]. For example, Ogawara et al.  reported that the PC adsorbed onto polystyrene NPs prolongs their circulation time, while Nagayama et al.  showed the PC on similar polystyrene NPs is responsible of increased clearance due to rapid recognition by the scavenger receptors and internalization by Kupffer cells. Examples of conflicting results can be found for other biological effects as well (e.g. cytotoxicity, targeting activity, etc.) [15-18].
Purification and analyses of PC-NP complexes
To ameliorate the difficulty interpreting the biological response observed by using NPs, researchers focalized the attention optimizing the procedure to characterize the PC around NPs. In particular, evaluating the overall quantity, density, thickness composition, relative abundance of each protein, protein binding affinity, and protein conformation. Firstly, PC-NP complexes were separated from the protein solution. This operation is not simple as the protein-NPs interaction is regulated by dynamic exchanges and equilibrium that are particularly sensitive to purification processes, altering the real contribution of the PC on the NP surface.
Commonly, sequential cycles of centrifugation/washing represent the most used method, because it is simple, suitable and gives reliable results [34,35]. However, multiple purification steps can alter the equilibrium of complexes and may lead to modification in corona compositions as previously explain . In particular, purification techniques adopted to isolate and to study the SC, which is governed by more dynamic exchanges, must be accurately selected and optimized in terms of operative conditions (time, temperature, stress, etc.). Moreover, due to high variability, in order to validate the data, several replicates must be collected and the results must be statistically relevant.
For this reason, the development of methodologies that minimize the number of purification steps of the PC-NP complexes in order to lower the possible impact of the process on their properties is broadly considered as an urgent issue . Beyond centrifugation, other techniques, summarized in Table 2, have also been applied to separate and study PC-NP complexes.
These techniques were successfully applied to isolate PC-NPs complexes in ex-vivo experiments, namely by incubating NPs in biological medium simulating relevant biological conditions. In total, only a few experiments reported reproducible results in terms of identification of PC isolated from plasma after in vivo administration. Actually, the idea is to exploit NP features and to adapt or combine different methodologies in function of the type of NPs. As an example, Sakulkhu et al.  demonstrated that the in vitro PC profile of polyvinyl alcohol-coated SPIONs differs from in vivo ones, after in vivo administration, by separating the NPs using a strong external magnetic field and therefore exploiting the unique magnetic properties of the particles. As another example, a combined approach was proposed by Hadjidemetriou et al.  to recover lipid-based NPs from the blood circulation of rodents after intravenous administration to investigate the in vivo PC formation as well as its evolution. In this case size exclusion chromatography followed by membrane ultrafiltration allowed the isolation of the PC-NP complex and to recover reproducible samples to identify the PC components.
After purification, several different approaches were proposed to characterize PC-NP complex in terms size, thickness, quantity, density, thickness composition, relative abundance of each protein, protein binding affinity and protein conformation (Table 3).
The interaction between proteins and the NPs is another relevant issue that could be addressed by different procedures. As an example, it is possible to consider physical changes in morphology, size and zeta potential of the complexes with respect to free NPs as proof of a PC-NP complex presence. Microscopical analyses using transmission electron microscopy (TEM) and in some cases, atomic force microscopy (AFM), could allow the visualization of differences in shape, density, surface and also dimensions of NPs before and after incubation while dynamic light scattering (DLS) rapidly identifies changes in dimensional distribution and poly-dispersity.
The interaction in terms of affinity, association and dissociation constant, and the stability of protein adsorbed were analyzed by using a plenty of techniques including isothermal titration calorimetry (ITC), circular dichroism, surface plasmon resonance, quartz crystal microbalance frequently combined together. Gel filtration has been proposed not only to separate the protein-NP complexes in incubation media as previously reported, but also to isolate proteins from the NP surfaces as well as to provide useful information on kinetic exchange rates for adsorbed proteins .
Besides proof of the PC-NPs complex presence, defining the PC represents a major topic: several researchers reported the use of electrophoresis as the preferred technology, while protein quantity determination was proposed to be characterized by mass spectrometry, in which protein samples were digested into small peptides and simply injected into the analysis instrument .
In summary, as evident, the identification and characterization of the PC cannot be obtained by a single analytical protocol, but different and complementary technologies are generally combined. In order to reach the highest level of quality and standardization in PC identification and characterization, the key point relies on the choice of the techniques in function of both the type of nanocarriers and on the analytical parameter to investigate. Using multiple characterization techniques is therefore crucial to analyze different aspects of the PC (i.e., presence of complex, composition of PC, reliability in in vivo conditions, etc.) and to get a better understanding of this biological entity. Remarkably, some techniques allow to detect the protein corona in situ (ITC), while other procedures require the detachment of bound proteins from the nanocarriers before measurements, thus still representing a controversial issue on PC analysis, since the adoption of purification methods may change equilibrium properties of the PC.
In recent years, the advance of nanomedicine as applied science in disease treatments highlighted a deeper need in understanding the interactions between nanocarriers and the biological environment aiming to improve their effectiveness and safety profiles. In this context, the study of the PC connected to any kind of NP drug carrier and its impact on both biodistribution and interaction with the target site is of extreme importance. Relevant information of the PC composition could also be exploited in sample screening at early research stages. This interest generated a wide number of attempted experiments, but at a deeper analysis of the results, even if remarkable, the lack of reproducibility and defined protocols in PC analysis still remain an urgent issue. In particular, as pointed out in this brief review, those data often obtained in vitro by simulating biological environments are affected by a high number of variables and seem not to be reliable and predictive for in vivo readouts and therefore their translatability. As also pointed out, numerous purification and investigation techniques were applied to evaluate and characterize the PC demonstrating that some technologies could really be useful in analysing some aspects of PC. Thus, in order to concretely exploit PC-NPs complexes data, the scientific path in this field will surely pass through an optimization of the protocols with a rational combination of the different techniques to finalize a systematic and more reproducible PC study approach.
Supported by MAECI grant (PI Tosi, Nanomedicine for BBB-crossing in CNS oncologic pathologies) and Emilia Romagna Region grant (Step-by-step screening: RCUP E56C18002000002).
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