The Protein Corona Conundrum: Exploring the Advantages and Drawbacks of its Presence around Amphiphilic Nanoparticles.

The success of targeted drug delivery systems still requires a detailed understanding about the biological consequences of self-developed biomolecular coronas around them, since this is the surface that interacts with living cells. Herein, we report the behavior of carbohydrate-decorated amphiphilic nanoparticles in a plasma environment with regard to the formation and biological consequences of the protein corona. Naked amphiphilic nanoparticles were produced through the self-assembly of azido-PEO900-docosanoate molecules, and the coupling of N-acetylglucosamine via click chemistry enabled the fabrication of the corresponding bioactive glyco-nanostructures. Light scattering measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography-mass spectrometry, and the Pierce BCA protein assay all confirmed the presence of protein coronas around the self-assembled nanoparticles, regardless of the presence of the sugar residues, although it reduces the amount of adsorbed proteins. The protein coronas were formed mainly by human serum albumin, complement proteins, apolipoproteins, immunoglobulins, and proteins involved in the coagulation cascade (fibrinogen and prothrombin). While the presence of these protein coronas significantly reduced cellular uptake of the amphiphilic assemblies, they also notably reduced the cytotoxic and hemolytic effects that result from the contact of the nanoparticles with living cells. Accordingly, we highlight that protein coronas should not always be treated as artifacts that have to be avoided because they can also provide beneficial effects.

Evidence of protein coronas around soft nanoparticles regardless of the chemical nature of the outer surface: structural features and biological consequences.

The formation of biomolecular coronas around nanoparticles as soon as they come in contact with biological media is nowadays well accepted. The self-developed biological outer surfaces can affect the targeting capability of the colloidal carriers as well as their cytotoxicity and cellular uptake behavior. In this framework, we explored the structural features and biological consequences of protein coronas around block copolymer assemblies consisting of a common pH-responsive core made by poly[2-(diisopropylamino) ethyl methacrylate] (PDPA) and hydrophilic shells of different chemical natures: zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) or highly hydrophilic poly(ethylene oxide) (PEO) and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA). We demonstrated the presence of ∼50 nm protein coronas around the nanoparticles regardless of the chemical nature of the polymeric shells. The thickness is understood as the sum of the soft and hard layers and it is the actual interface seen by the cells. Although the soft corona composition is difficult to determine because the proteins are loosely bound to the outer surface of the assemblies, the tightly bound proteins (hard corona) could be identified and quantified. The compositional analysis of the hard corona demonstrated that human serum albumin (HSA), immunoglobulin G (IgG) and fibrinogen are the main components of the protein coronas, and serotransferrin is present particularly in the protein corona of the zwitterionic-stabilized assemblies. The protein coronas substantially reduce the cellular uptake of the colloidal particles due to their increased size and the presence of HSA which is known to reduce nanoparticle-cell adhesion. On the other hand, their existence also reduces the levels of cytotoxicity of the polymeric assemblies, highlighting that protein coronas should not be always understood as artifacts that need to be eliminated due to their positive outputs.

Positively Selected Sites at HCMV gB Furin Processing Region and Their Effects in Cleavage Efficiency.

Human cytomegalovirus is a ubiquitous infectious agent that affects mainly immunosuppressed, fetuses, and newborns. The virus has several polymorphic regions, in particular in the envelope glycoproteins. The UL55 gene encodes the glycoprotein B that has a variable region, containing a furin cleavage site and according to the variability different genotypes are characterized. Here we investigated variability and existence of selective pressure on the UL55 variable region containing the furin cleavage site in 213 clinical sequences from patients worldwide. We showed the occurrence of positive selective pressure on gB codons 461 and 462, near the furin cleavage site. Cleavage analysis of synthesized peptides demonstrated that most mutations confer better cleavage by furin, suggesting that evolution is acting in order to increase the efficiency cleavage and supporting the hypothesis that gB processing is important in the host. We also demonstrated that peptides containing sequences, that characterize genotypes gB2 and 3, are differentially cleaved by furin. Our data demonstrate for the first time that variability in the cleavage site is related to degree of gB processing by furin.

Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades

Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties.