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.

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.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

Sweetness Reduces Cytotoxicity and Enables Faster Cellular Uptake of Sub-30 nm Amphiphilic Nanoparticles.

Glycoconjugates are versatile entities used for the manufacturing of targeted drug delivery nanocontainers because of their outstanding capability to bind to lectins, which are proteins that can be found overexpressed in the membranes of unhealthy cells. The assisted attachment to pathological cells can further enable a more efficient intracellular delivery of loaded active agents, thereby reducing side effects that commonly compromise chemotherapies. In this framework, azide-terminated polyethylene oxide (PEO) chains coupled to a 22-carbon chain were synthesized (azide-PEO900-docosanoate). The resulting amphiphile was further functionalized by introducing different sugar moieties to the PEO chains via the click chemistry approach. Sub-30 nm, negatively charged, and spherical nanoparticles were prepared in water by self-assembly of the synthesized molecules using the straightforward nanoprecipitation protocol. The produced entities do not induce hemolysis in red blood cells at c ≤ 200 µg mL-1, and they are not cytotoxic to healthy cells [telomerase immortalized rhesus fibroblasts (Telo-RF)] at c ≤ 50 µg mL-1. The sugar-decorated nanoparticles are less cytotoxic compared with their naked counterparts at the concentration range assessed. The kinetics of cellular uptake of both entities into normal (Telo-RF) and tumor (HeLa) cells were monitored via fluorescence microscopy and flow cytometry. The nanoparticles are internalized faster in cancer cells than in normal cells, regardless of functionalization. Moreover, the functionalized nanoparticles are internalized faster in HeLa cells, while the reverse was observed in healthy Telo-RF cells. The distinct surface characteristics of the assemblies create an opportunity to expedite the uptake of nanoparticles particularly by tumor cells, and this accordingly can lead to a more effective intracellular delivery of therapeutic molecules loaded into nanoparticle's reservoirs.

Nanoparticle-Cell Interactions: Surface Chemistry Effects on the Cellular Uptake of Biocompatible Block Copolymer Assemblies.

The development of nanovehicles for intracellular drug delivery is strongly bound to the understating and control of nanoparticles cellular uptake process, which in turn is governed by surface chemistry. In this study, we explored the synthesis, characterization, and cellular uptake of block copolymer assemblies consisting of a pH-responsive poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) core stabilized by three different biocompatible hydrophilic shells (a zwitterionic type poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) layer, a highly hydrated poly(ethylene oxide) (PEO) layer with stealth effect, and an also proven nontoxic and nonimmunogenic poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) layer). All particles had a spherical core-shell structure. The largest particles with the thickest hydrophilic stabilizing shell obtained from PMPC40-b-PDPA70 were internalized to a higher level than those smaller in size and stabilized by PEO or PHPMA and produced from PEO122-b-PDPA43 or PHPMA64-b-PDPA72, respectively. Such a behavior was confirmed among different cell lines, with assemblies being internalized to a higher degree in cancer (HeLa) as compared to healthy (Telo-RF) cells. This fact was mainly attributed to the stronger binding of PMPC to cell membranes. Therefore, cellular uptake of nanoparticles at the sub-100 nm size range may be chiefly governed by the chemical nature of the stabilizing layer rather than particles size and/or shell thickness.

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.

Influence of Structural Features on the Cellular Uptake Behavior of Non-Targeted Polyester-Based Nanocarriers.

The development of delivery systems efficiently uptaken by cells is of due importance since sites of drug action are generally localized in subcellular compartments. Herein, naked and core-shell polymeric nanoparticles (NPs) have been produced from poly(lactic-co-glycolic acid)-PLGA, poly(ethylene oxide)-b-poly(ε-caprolactone)-PEO-b-PCL, and poly(ethylene oxide)-b-poly(lactic acid)-PEO-b-PLA. The nanostructures are characterized and the cellular uptake behavior is evaluated. The data evidence that cellular uptake is enhanced as the length of the hydrophilic PEO-stabilizing shell reduces and that high negative surface charge restricts cellular uptake. Furthermore, NPs of higher degree of hydrophobicity (PEO-b-PCL) are more efficiently internalized as compared to PEO-b-PLA NPs. Accordingly, taking into account our recent published results and the findings of the current investigation, there should be a compromise regarding protein fouling and cellular uptake as resistance to nonspecific protein adsorption and enhanced cellular uptake are respectively directly and inversely related to the length of the PEO-stabilizing shell.

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties.

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells.

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells.