Journal: ACS Biomaterials Science & Engineering

Article Title: Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells

doi: 10.1021/acsbiomaterials.1c00804

Figure Lengend Snippet: Effect of free transferrin on nanoparticle uptake in TRP3 liver endothelium cells. (a) Uptake of 30 μg mL –1 corona-coated nanoparticle complexes formed on 200 nm SiO 2 –NH 2 in full human plasma in the presence of increasing concentrations of human transferrin in serum-free medium after a 4 h exposure. (b) Uptake of 10 μg mL –1 Alexa Fluor 546 fluorescently labeled transferrin in the presence of increasing concentrations of the isolated corona-coated nanoparticle complexes in serum-free medium. (c) Uptake of corona-coated nanoparticle complexes in TRP3 cells after silencing the expression of transferrin receptor 1 (TFR1). TFR1 expression was silenced as described in the section, then cells were exposed for 4 h to 30 μg mL –1 nanoparticle-corona complexes in serum-free medium or in the presence of 1 mg mL –1 human transferrin. The results of three independent experiments are shown, together with their average indicated with a line. One of the 3 repeated experiments of panels a and b was performed using a different batch of nanoparticles (see Figure S3, Supporting Information for more details). Nevertheless, as shown in these panels, the results were highly reproducible. The competition experiments showed that free transferrin increased nanoparticle uptake instead of competing with it, while the uptake of transferrin decreased when corona-coated nanoparticle complexes were added. A Mann–Whitney test was applied to compare the uptake level in serum-free conditions (0 μg mL –1 competitor) and when (a) transferrin or (b) the corona-coated nanoparticles were added at the highest concentration. For the results in part c, a Mann–Whitney test was applied to compare uptake levels after addition of transferrin. p ≤ 0.05 was considered significant (indicated with an asterisk).

Article Snippet: Green fluorescently labeled silica nanoparticles (maximum excitation 485 nm and emission 510 nm) of 100 and 200 nm diameter, and with a plain, amino-modified or carboxylated surface (SiO 2 , SiO 2 –NH 2 , and SiO 2 –COOH, respectively), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany).

Techniques: Clinical Proteomics, Labeling, Isolation, Expressing, MANN-WHITNEY, Concentration Assay

Journal: ACS Biomaterials Science & Engineering

Article Title: Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells

doi: 10.1021/acsbiomaterials.1c00804

Figure Lengend Snippet: Uptake of single protein corona-coated nanoparticles in liver (a) and brain endothelium (b). The 200 nm SiO 2 –NH 2 were coated with 15 μg mL –1 human plasma, HRG, transferrin, HSA, or alpha-1 antitrypsin as described in the section, and 30 μg mL –1 corona-coated nanoparticles were added to cells for 4 h in serum-free medium. As additional controls, the uptake in serum-free medium of 30 μg mL –1 bare nanoparticles (bare NP) and nanoparticles coated with full human plasma corona prepared as described in the section (full human plasma) was also measured. The results of three independent experiments are shown, together with their average indicated with a line. A Kruskal–Wallis test was used to compare the different groups and indicated significant differences in both panels. A Mann–Whitney test with Bonferroni correction for multiple testing was applied to compare the uptake level of single protein corona-coated nanoparticles to the uptake of bare nanoparticles (bare NP) or nanoparticles coated with 15 μg mL –1 human plasma (human plasma). p ≤ 0.05 was considered significant (indicated with an asterisk).

Article Snippet: Green fluorescently labeled silica nanoparticles (maximum excitation 485 nm and emission 510 nm) of 100 and 200 nm diameter, and with a plain, amino-modified or carboxylated surface (SiO 2 , SiO 2 –NH 2 , and SiO 2 –COOH, respectively), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: ACS Biomaterials Science & Engineering

Article Title: Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells

doi: 10.1021/acsbiomaterials.1c00804

Figure Lengend Snippet: Uptake kinetics of the corona-coated nanoparticles isolated from full human plasma. Brain (a and e), lung (b and f), liver (c and g), and kidney endothelium (d and h) were exposed to 50 μg mL –1 of 100 nm (a–d) or 30 μg mL –1 of 200 nm (e–h) corona-coated SiO 2 , SiO 2 –NH 2 , or SiO 2 –COOH in serum-free medium, isolated from full human plasma as described in the section. The results show the median cell fluorescence intensity of two replicate samples, together with a line that passes through their average. A linear regression two-tailed Student’s t -test was applied to compare the uptake of the different corona-coated nanoparticles. Statistically significant differences up to a 7 h uptake (indicated by an asterisk) are observed for all cell types on 100 nm nanoparticles for SiO 2 –COOH as compared to the other functionalizations and on 200 nm nanoparticles for SiO 2 –NH 2 . p ≤ 0.05 was considered significant.

Article Snippet: Green fluorescently labeled silica nanoparticles (maximum excitation 485 nm and emission 510 nm) of 100 and 200 nm diameter, and with a plain, amino-modified or carboxylated surface (SiO 2 , SiO 2 –NH 2 , and SiO 2 –COOH, respectively), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany).

Techniques: Isolation, Clinical Proteomics, Fluorescence, Two Tailed Test

Journal: ACS Biomaterials Science & Engineering

Article Title: Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells

doi: 10.1021/acsbiomaterials.1c00804

Figure Lengend Snippet: Characterization of the corona formed on 100 and 200 nm SiO 2 (plain), SiO 2 –NH 2 (NH 2 ), or SiO 2 –COOH (COOH) in full human plasma. SDS-PAGE gel image of the proteins recovered on nanoparticle-corona complexes of 100 nm (a) or 200 nm (b) silica in full human plasma. The corona formed on all silica nanoparticles was prepared and isolated as described in the section. The gel shows that different bands were present in the corona formed on the different silica nanoparticles. M: molecular weight size marker. Relative abundance (RPA%, see the section for details) of the major protein groups identified by mass spectrometry in full human plasma and in the protein corona formed on the different silica nanoparticles (c). Venn diagram of the total amount of proteins identified by mass spectrometry in the nanoparticle-corona complexes formed in full human plasma (d). List of the top 20 most abundant corona proteins and their RPA (%) on the indicated silica nanoparticles, as measured by mass spectrometry (e). Proteins are ordered alphabetically. Different colors are used for the different protein families, and the spot size indicates their RPA (%).

Article Snippet: Green fluorescently labeled silica nanoparticles (maximum excitation 485 nm and emission 510 nm) of 100 and 200 nm diameter, and with a plain, amino-modified or carboxylated surface (SiO 2 , SiO 2 –NH 2 , and SiO 2 –COOH, respectively), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany).

Techniques: Clinical Proteomics, SDS Page, Isolation, Molecular Weight, Marker, Mass Spectrometry

Journal: ACS Biomaterials Science & Engineering

Article Title: Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells

doi: 10.1021/acsbiomaterials.1c00804

Figure Lengend Snippet: Correlation between nanoparticle uptake in brain, lung, liver, and kidney endothelium after 5h exposure and the relative protein abundance of adsorbed corona proteins for all six silica nanoparticles tested. The uptake level of the nanoparticle-corona complexes formed on 100 (a) and 200 nm (b) silica nanoparticles in full human serum after 5 h. The results show the median cell fluorescence intensity of two replicate samples, together with their average indicated with a line. (c) Corona proteins correlating with uptake. The table shows the results of the correlation analysis between the 5-h nanoparticle uptake in brain, lung, liver, and kidney endothelium and the relative protein abundance of adsorbed corona proteins, performed as described in the section. Positive correlation coefficients ( r ) ≥ 0.6 are shaded in light gray, and negative correlation coefficients ( r ) ≤ 0.6 are shaded in dark gray.

Article Snippet: Green fluorescently labeled silica nanoparticles (maximum excitation 485 nm and emission 510 nm) of 100 and 200 nm diameter, and with a plain, amino-modified or carboxylated surface (SiO 2 , SiO 2 –NH 2 , and SiO 2 –COOH, respectively), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany).

Techniques: Quantitative Proteomics, Fluorescence

Journal: ACS Biomaterials Science & Engineering

Article Title: Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells

doi: 10.1021/acsbiomaterials.1c00804

Figure Lengend Snippet: Scheme illustrating the proposed mechanism of uptake for 200 nm SiO 2 –NH 2 nanoparticles in liver endothelium. The thickness of the arrows represents uptake efficiency. (a) Uptake of particles with a human plasma corona is lower than for bare particles, but the same effect is obtained with a corona made by HRG alone. (b) Particles with a human plasma corona enter cells via multiple receptors including the transferrin (Tf) receptor. However, if free Tf is added (c), it competes with the particles for the Tf receptors, and the particles are displaced to a different receptor which has higher uptake efficiency, leading to a higher uptake.

Article Snippet: Green fluorescently labeled silica nanoparticles (maximum excitation 485 nm and emission 510 nm) of 100 and 200 nm diameter, and with a plain, amino-modified or carboxylated surface (SiO 2 , SiO 2 –NH 2 , and SiO 2 –COOH, respectively), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany).

Techniques: Clinical Proteomics

Journal: Scientific Reports

Article Title: Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

doi: 10.1038/s41598-021-83106-2

Figure Lengend Snippet: Structure and function of omphalotin biosynthetic gene cluster and homologous clusters in the basidiomycetes O. olearius , D. bispora and L. edodes. ( A ) Schematic representation of the borosin biosynthetic gene clusters in the basidiomycetes O. olearius (VT 653.13, scaffold _ 169), L. edodes (Le(Bin) 0899 ss11, scaffold_10) and D. bispora (CBS 962.96, scaffold_621). All three clusters code for a precursor (methyltransferase) protein, referred to as OphMA, LedMA and DbiMA1 respectively, and a prolyloligopeptidase, referred to as OphP, LedP and DbiP, respectively. The DNA scaffolds and filtered gene models were taken from https://mycocosm.jgi.doe.gov/ . Double slashes (//) indicate that the sequence of the DNA scaffold continues beyond this position. ( B ) Biosynthesis scheme of omphalotin A and closely related peptides, lentinulin A and dendrothelin A as a combined action of the precursor (methyltransferase) protein and the prolyloligopeptidase. ( C ) Alignment of the C-termini of the precursor (methyltransferase) proteins from O. olearius (OphMA), L. edodes (LedMA) and D. bispora (DbiMA1). Residues differing from the omphalotin core peptide are shown in red. The indicated methylation patterns (green fill) were previously determined by heterologous expression of the respective cDNAs in E. coli , , . CRS = C-terminal recognition sequence.

Article Snippet: Omphalotus olearius (strain DSM3398) was ordered from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ; Leibniz Institute, Germany), Dendrothele bispora (strain CBS 962.96) from the Westerdijk Fungal Biodiversity Institute strain collection (The Netherlands), and Lentinula edodes (strain 4312 from Sylvan, China) was a gift from a local mushroom farm (Fine Funghi AG, Gossau ZH, Switzerland).

Techniques: Sequencing, Methylation, Expressing

Journal: Scientific Reports

Article Title: Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

doi: 10.1038/s41598-021-83106-2

Figure Lengend Snippet: LC–MS analysis of backbone N-methylated peptides extracted from mushrooms O. olearius , L. edodes and D. bispora . ( A – C ) Ion chromatograms depicting some of the peptides species produced by 28 days liquid cultures of the respective fungi. In case of O. olearius ( A ) and D. bispora ( B ), both backbone-N-methylated macrocyclic peptides (M + H + ) and additionally oxidized macrocyclic peptides (M + nO + H) + thereof were observed in the culture supernatant. In case of the L. edodes liquid culture ( C ) , oxidized cyclic peptides were detected in the fungal mycelium. (M + H 2 O + H) + represents the linear form of the macrocyclic peptide. ( D ) Chemical structures of omphalotin A, lentinulin A and dendrothelin A. Residues different from omphalotin A are underlined. Backbone N-methylation is indicated by green circles.

Article Snippet: Omphalotus olearius (strain DSM3398) was ordered from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ; Leibniz Institute, Germany), Dendrothele bispora (strain CBS 962.96) from the Westerdijk Fungal Biodiversity Institute strain collection (The Netherlands), and Lentinula edodes (strain 4312 from Sylvan, China) was a gift from a local mushroom farm (Fine Funghi AG, Gossau ZH, Switzerland).

Techniques: Liquid Chromatography with Mass Spectroscopy, Methylation, Produced

Journal: Polymers

Article Title: Surface Modification of Wood Flour via ARGET ATRP and Its Application as Filler in Thermoplastics

doi: 10.3390/polym10040354

Figure Lengend Snippet: Fluorescence image of stained wood flour.

Article Snippet: Dried wood flour containing thiol groups was immersed in a solution of 32 μg·mL −1 fluorescence dye (Abberior LIVE 510) in PBS (phosphate-buffered saline, pH 7) and shaken overnight in the dark.

Techniques: Fluorescence, Staining