Fetal Bovine Serum Proteins Research Articles

Surface property and in vitro toxicity effect of insoluble particles given by protein corona: Implication for PM cytotoxicity assessment

In vitro toxicological assessment helps explore key fractions of particulate matter (PM) in association with the toxic mechanism. Previous studies mainly discussed the toxicity effects of the water-soluble and organic-soluble fractions of PM. However, the toxicity of insoluble fractions is relatively poorly understood, and the adsorption of proteins is rarely considered. In this work, the formation of protein corona on the surface of insoluble particles during incubation in a culture medium was investigated. It was found that highly abundant proteins in fetal bovine serum were the main components of the protein corona. The adsorbed proteins increased the dispersion stability of insoluble particles. Meanwhile, the leaching concentrations of some metal elements (e.g., Cu, Zn, and Pb) from PM increased in the presence of proteins. The toxicity effects and potential mechanisms of the PM insoluble particle–protein corona complex on macrophage cells RAW264.7 were discussed. The results revealed that the PM insoluble particle–protein corona complex could influence the phagosome pathway in RAW264.7 cells. Thus, it promoted the intracellular reactive oxygen species generation and induced a greater degree of cell differentiation, significantly altering cell morphology. Consequently, this work sheds new light on the combination of insoluble particles and protein corona in terms of PM cytotoxicity assessment.

Polyethylene glycol precipitation is an efficient method to obtain extracellular vesicle-depleted fetal bovine serum.

Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination.

Effect of the oxygenic groups on activated carbon on its hemocompatibility

In this research, the effect of the oxygenic groups on activated carbon on its hemocompatibility was studied by liquid-phase oxidation to introduce oxygenic groups on its surface and subsequent heat treatment under a nitrogen environment to remove these groups. Hemocompatibility was assessed through coagulation, hemolysis, platelet adhesion, and protein adsorption using rabbit blood samples. Results showed that an increasing presence of oxygenic groups improved hemocompatibility, evidenced by enhanced coagulation, reduced hemolysis, better platelet adhesion, and decreased fetal bovine serum protein adsorption. Conversely, the removal of oxygenic groups diminished hemocompatibility, except for coagulation when groups were removed at 250 ℃ for 15 min. Therefore, this research presents a promising route to enhance the hemocompatibility of activated carbon, offering insights into surface modification for improved biomaterial design.

LSPR-Based Aptasensor for Rapid Urinary Detection of NT-proBNP.

N-terminal pro-brain natriuretic peptide (NT-proBNP) is a myocardial stress biomarker that can be found in serum or plasma, saliva, and urine in the context of cardiovascular disease. In this study, we developed a rapid (~25 min) and straightforward localized surface plasmon resonance (LSPR)-based assay for detecting NT-proBNP in urine. The assay employs citrate-capped gold nanoparticles (AuNPs) and an aptamer specific for NT-proBNP, which initially interacts with NT-proBNP. The remaining unbound aptamer then interacts with the AuNPs, and the addition of NaCl induces the aggregation of the unprotected AuNPs, resulting in a decrease in absorbance at the LSPR band (A521) and an increase in absorbance at 750 nm (A750). The concentration of NT-proBNP showed a linear correlation with the aggregation ratio (A521/A750), and the assay demonstrated a limit of detection (LOD) of 0.303 µg·L-1 and a detection range of 0.566-8 µg·L-1. However, the presence of sulfur-containing proteins in saliva and fetal bovine serum hindered the detection of NT-proBNP in these biofluids. Nevertheless, the assay successfully detected NT-proBNP in diluted urine with an LOD of 0.417 µg·L-1 and a detection range of 0.589-6 µg·L-1. The observed values in urine samples from preterm infants with cardiovascular disease fell within this range, indicating the potential clinical relevance of the assay. The recovery percentages ranged from 92.3 to 116.3%. Overall, our findings suggest that the LSPR-based assay for NT-proBNP detection in urine can be a valuable tool for the diagnosis and treatment of cardiovascular disease.

Predicting the interaction of nanoparticles with proteins using lab automation and machine learning.

Nanoparticles in biological fluids interact with proteins forming a “corona” of proteins adsorbed on the nanoparticle surface. Subsequent biological interactions and responses are determined by the protein corona rather than the bare nanoparticle. The development of more effective nanomedicines and nanosensors requires a better understanding of the initial protein-nanoparticle interaction. We have developed an automated workflow, using a liquid handling robot coupled with low-cost proteomics, to generate and characterize protein coronas in a 96-well format. We have used this approach to characterize the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200 nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. Current research is aimed at using random forest regression and a suite of nanoparticles (50 nm, 150 nm, 300 nm; −50 mV to +40 mV) to determine what nanoparticle surface properties lead to the adsorption of specific proteins.

Concentration and composition of the protein corona as a function of incubation time and serum concentration: an automated approach to the protein corona.

Nanoparticles in contact with proteins form a "corona" of proteins adsorbed on the nanoparticle surface. Subsequent biological responses are then mediated by the adsorbed proteins rather than the bare nanoparticles. The use of nanoparticles as nanomedicines and biosensors would be greatly improved if researchers were able to predict which specific proteins will adsorb on a nanoparticle surface. We use a recently developed automated workflow with a liquid handling robot and low-cost proteomics to determine the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. In addition to these findings, we describe two methodological advances to help reduce the cost associated with protein corona experiments. We have automated the digest step necessary for proteomics and measured the variability between triplicate samples at each stage of the proteomics experiments. Overall, these results demonstrate the importance of understanding the multiple parameters that influence corona formation, provide new tools for corona characterization, and advance bioanalytical research in nanomaterials.

Nano-Sized Polyelectrolyte Complexes Formed between Poly(vinyl benzyl trimethyl ammonium chloride) and Insulin

Novel biohybrid homo-polyelectrolyte-based nanocarriers were formed by the complexation of insulin (INS) with a biocompatible and cationic polyelectrolyte, namely, poly(vinyl benzyl trimethylammonium chloride) (PVBTMAC). According to light-scattering techniques, the hydrophilic PVBTMAC homo-polyelectrolyte forms single chains in aqueous media. The resulting biohybrid PVBTMAC/INS nanocarriers were formed via electrostatic co-assembly. The effects of polyelectrolyte structure and content on the characteristics of the formed PVBTMAC/INS complexes were studied. A significant aggregation tendency of the PVBTMAC/INS complexes was observed, based on the physicochemical results, especially at high protein concentration, corroborating the effective electrostatic interaction of INS with the cationic polyelectrolyte. The physicochemical properties of the formed PVBTMAC/INS nanocarriers depended on the concentration of the stock polymer and INS solutions. A neat PVBTMAC homo-polymer and PVBTMAC/INS nanocarriers demonstrated good serum stability in the presence of fetal bovine serum (FBS) proteins. Fluorescence spectroscopy (FS) studies revealed no INS conformational changes after its complexation with the cationic PVBTMAC polyelectrolyte. The obtained PVBTMAC/INS complexes demonstrated considerable and promising characteristics for potential use as insulin delivery systems.

Probing the Role of Charged Functional Groups on Nanoparticles Grafted with Polyglycerol in Protein Adsorption and Cellular Uptake

AbstractIn biofluids, charged functional groups on the surface of nanoparticles (NPs) interact with cells through the protein corona. However, the cascade effects of charged groups on corona formation and cellular uptake remain unclear. Herein, carboxy, sulfate, and amino groups are quantitatively introduced at the periphery of polyglycerol (PG)‐grafted nanodiamond and superparamagnetic iron oxide NP to probe their roles in corona formation and cellular uptake. The uptake efficiency and intracellular aggregation state of NPs are revealed to correlate with protein affinity of the charged groups; sulfate at lower density and carboxylate exhibit no affinity to proteins, inducing negligible or no cellular uptake. In contrast, sulfate at higher density and ammonium associate with fetal bovine serum proteins to alter the aggregation state of the internalized NPs. It is further demonstrated that the distinct protein corona profiles on NP‐PG‐OSO3− and NP‐PG‐NH3+ surfaces dictate their uptake mechanism. The protein corona of NP‐PG‐OSO3− suppresses cellular uptake via downregulation of macropinocytosis and clathrin‐mediated endocytosis, whereas that of NP‐PG‐NH3+ enhances uptake through upregulation of macropinocytosis and caveolae‐mediated endocytosis. This study clarifies the elusive role of the charged groups in protein adsorption and cellular uptake, which sheds light on NP design for controlled cellular uptake and theranostics in nanomedicine.

A Simple Method for In-Depth Proteome Analysis of Mammalian Cell Culture Conditioned Media Containing Fetal Bovine Serum.

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

Developing in vitro models to assess fish gill excretion of emerging contaminants.

Advances in analytical methods have enabled the detection of emerging contaminants at ever lower concentrations in freshwaters. However, such measurements need to be linked to effect-based assays to identify risks. The bioconcentration factor (BCF) forms part of a chemical's environmental risk assessment (ERA), and current regulatory testing guidelines to calculate fish BCFs use hundreds of fish per chemical. Due to ethical concerns a reduction in the numbers of animals used is desired, and there is a need to identify in vitro or in silico alternatives which meet regulatory acceptance. This study describes the successful demonstration of a FIsh Gill Cell culture System (FIGCS) to assess an often overlooked parameter in pharmacokinetics: the excretion of drugs across the gill. The FIGCS tolerates the application of natural waters on its apical surface, mimicking the situation of the live fish, and thus in combination with advanced analytical methods, offers an opportunity to take lab-based testing used for ERA, such as compound uptake, biotransformation or excretion directly into field for validation with natural waters. Here we used the basic drug propranolol and the acidic ibuprofen as a demonstration of the FIGCS utility in three separate experiments. Excretion across the apical membrane showed saturation kinetics, suggesting the involvement of carrier-mediated processes. Both propranolol and ibuprofen were excreted across the epithelium from the media (internal blood equivalent) to the water, with ibuprofen excretion being considerably slower than propranolol excretion. Further studies indicate that ibuprofen may be complexing with fetal bovine serum (FBS) reducing bioavailability; in contrast propranolol efflux rate was unaffected, indicating that drugs behave differently in the presence of FBS and other plasma proteins. A key issue in future ERA is to better understand the effects of mixtures of different pollutant classes found in environmental samples, and this model offers an ethical path to do this.

Effects of Surface Chemistry and Topology on the Kinesin-Driven Motility of Microtubule Shuttles.

Nanoscale transport using the kinesin-microtubule system has been successfully used in applications ranging from self-assembly, to biosensing, to biocomputation. Realization of such applications necessitates robust microtubule motility particularly in the presence of complex sample matrices that can affect the interactions of the motors with the surface and the transport function. In the present work, we explored how the chemical nature and nanoscale topology of various surfaces affected kinesin-microtubule transport. Specifically, we characterized microtubule motility on three distinct interfaces: (i) surfaces modified with self-assembled monolayers (SAMs) displaying three different terminal groups, (ii) SAM-modified surfaces with adsorbed fetal bovine serum (FBS) proteins, and (iii) surfaces where the FBS layer was silicified to preserve an underlying surface topology. The composition and topology of each surface was confirmed with a number of techniques including X-ray photoelectron spectroscopy (XPS), water contact angle, atomic force microscopy (AFM), and scanning electron microscopy (SEM). The majority of surfaces, with the exception of those with the hydrophobic SAM, supported gliding motility consistent with the glass control. Differences in the displacement, velocity, and trajectory of the leading tip of the microtubule were observed in relation to the specific surface chemistry and, to a lesser extent, the nanoscale topology of the different substrates. Overall, this work broadens our understanding of how surface functionality and topology affect kinesin-based transport and provides valuable insights regarding future development of biosensing and probing applications that rely on biomolecular transport.

Compromised Autophagic Effect of Polystyrene Nanoplastics Mediated by Protein Corona Was Recovered after Lysosomal Degradation of Corona.

The adverse biological and ecological consequences of plastic debris have become a serious problem worldwide. Evidences have uncovered the accumulation of nanoplastics (NPs) in organisms. In a complex biological environment, proteins are prone to adsorbed onto the NPs' surface and form a protein corona layer, which mediates the interaction of NPs with cells. Here, we discovered the interaction of polystyrene (PS) NPs with protein fetal bovine serum (FBS) and altered cytotoxic effects. Mechanistically, prefabricated FBS protein corona mediated the relief of autophagic flux blockage, autophagosomes accumulation, and lysosomal damage in RAW264.7 cells caused by PS NPs. Using an individual fluorescent protein bovine serum albumin (BSA) as a corona surrogate, we demonstrated that coronal BSA remains, at least partially, on the surface of PS NPs during the initial stage of internalization and protects cell membrane from PS NPs-induced damage. However, along with the degradation of corona in lysosomes, reappearance of cytotoxicity was observed. Herein, we provided a proof of principle of the manipulation of corona on NPs' toxicity and we expect the result will promote the further safety assessment of NPs.

Polyglycerol Grafting Shields Nanoparticles from Protein Corona Formation to Avoid Macrophage Uptake.

Upon contact with biofluids, proteins are quickly adsorbed onto the nanoparticle (NP) surface to form a protein corona, which initiates the opsonization and facilitates the rapid clearance of the NP by macrophage uptake. Although polyethylene glycol (PEG) functionalization has been the standard approach to evade macrophage uptake by reducing protein adsorption, it cannot fully eliminate nonspecific uptake. Herein, polyglycerol (PG) grafting is demonstrated as a better alternative to PEG. NPs of various size and material were grafted with PG and PEG at 30, 20, and 10 wt % contents by controlling the reaction conditions, and the resulting NP-PG and NP-PEG were characterized qualitatively by IR spectroscopy and quantitatively by thermogravimetric analysis. Their resistivity to adsorption of the proteins in fetal bovine serum and human plasma were compared by polyacrylamide gel electrophoresis, bicinchoninic acid assay, and liquid chromatography-tandem mass spectrometry, giving a consistent conclusion that PG shields protein adsorption more efficiently than does PEG. The macrophage uptake was assayed by transmission electron microscopy and by extinction spectroscopy or inductively coupled plasma mass spectrometry, revealing that PG avoids macrophage uptake more efficiently than does PEG. In particular, a NP coated with PG at 30 wt % (NP-PG-h) prevents corona formation almost completely, regardless of NP size and core material, leading to the complete evasion of macrophage uptake. Our findings demonstrate that PG grafting is a promising strategy in nanomedicine to improve anti-biofouling property and stealth efficiency in nanoformulations.

Structural and Biological Analysis of Mesoporous Lanthanum Doped βTCP For Potential Use as Bone Graft Material

In this study, mesoporous particles of β-tricalcium phosphate (βTCP, βCa3(PO4)2) and lanthanum (La) doped βTCP were synthesized using wet precipitation method attached with microwave refluxing system. The obtained materials were characterized and analysed using different sort of techniques such; X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Scanning electron microscope (SEM), Inductively coupled plasma optical emission spectroscopy (ICP-OES), Brunauer–Emmett–Teller (BET) and helium pycnometer. With incorporation of La3+ ions, relevant expansion in lattice parameters of βTCP crystal was observed along with inhibition growth rate was observed. βTCP and La- βTCP materials were mesoporous in nature, pore diameter and pore volume were expanded with incorporation of La3+ ions. The loading /release on βTCP and La- βTCP particles surfaces were evaluated using fetal bovine serum (FBS) proteins. In vitro cell culture studies were performed with human osteosarcoma cell line (Saos-2). The outcomes approved that all La-βTCP materials were cytocompatible and strict dose dependent effect of La3+ ions was observed on cell viability and alkaline phosphatase (ALP) activity. These results strongly suggest that La-βTCP materials have a potential application in bone tissue engineering.

The Inflammatory Lipid, 2‐Chlorohexadecanal: Cell Toxicity and Potential Scavenger Protection

Hypochlorous acid is produced by either leukocyte myeloperoxidase activity or from chlorine gas reactions with water. 2‐Chlorofatty aldehydes (2‐ClFALD) are: 1) formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and 2) produced following leukocyte activation as well in the lung of mice exposed to chlorine gas. The biological role of 2‐ClFALD is largely unknown. Recently, our lab showed the electrophilic α‐chlorinated carbon of the 2‐ClFALD molecular species, 2‐chlorohexadecanal (2‐ClHDA) is targeted by nucleophilic attack of the cysteinyl‐sulfhydryl group of glutathione. Here, we synthesized an alkyne analog of 2‐ClHDA (2‐ClHDyA) to demonstrate 2‐ClHDyA protein modification in RAW 264.7 cells using click chemistry strategies. Furthermore, confocal microscopy showed that 2‐ClHDyA co‐localizes to the endoplasmic reticulum, Golgi, and mitochondria. Additionally, diffuse distribution of 2‐ClHDyA suggested cytoplasmic protein modification. The intensity of the fluorescence signal of 2‐ClHDyA increased in a dose‐dependent manner suggesting increased protein modification. Cell viability/metabolic activity assessed by 3‐(4,5‐Dimethyl‐2‐Thiazolyl)‐2,5‐Diphenyl‐2H‐Tetrazolium Bromide (MTT) test revealed that 2‐ClHDA reduced RAW 264.7 cell viability in a dose‐dependent manner. Cell viability was further reduced in RAW 264.7 cells treated with 2‐ClHDA in DMEM with Fetal Bovine Serum (FBS) supplementation. Click chemistry showed 2‐ClHDyA modified FBS proteins. These observations suggested 2‐ClFALD modified proteins might have accounted for cell death via cell signaling outside or inside the cell. Next, we investigated traditional aldehyde sequesterers including N‐acetyl cysteine (NAC), carnosine, and aminoguanidine to identify potential protectors that would prevent cellular 2‐ClFALD‐protein interaction resulting in reduced toxicity caused by 2‐ClFALD. Among tested sequesterers, NAC proved to be an effective sequesterer of 2‐ClHDA. NAC prevented 2‐ClHDyA interaction with FBS proteins and rescued cell death significantly. Next, we characterized the conjugate of 2‐ClHDA and NAC using direct infusion electrospray mass spectrometry. The conjugate appeared to be a result of a nucleophilic substitution reaction where nucleophilic sulfur of NAC attacked the alpha carbon of 2‐ClHDA leading to the release of chloride ion. These studies demonstrate that the interaction of 2‐ClFALD with cellular proteins with free sulfhydryl groups cause toxicity and the interaction of 2‐ClFALD with N‐acetyl cysteine may protect against toxicity caused by 2‐ClFALD.Support or Funding InformationNIH grants: R01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697 and U01 ES028182

Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Lowering protein fouling by rational processing of fluorine-free hydrophobic coatings

Protein fouling has a tremendous economic impact and thus requires persistent endeavors directed towards designing surfaces that are able to suppress this phenomenon. One approach is to use multifunctional protective coatings and reduce protein adhesion. While it is commonly accepted that hydrophilic surfaces repel proteins, conflicting data have lately concerned their hydrophobic counterparts. Here we investigate the potential in reducing protein adhesion of hydrophobic surfaces, glass (GS) and stainless steel (SS), modified by a two-step inorganic and a multi-step organic coatings. The inorganic coating derives from methyltrimethoxysilane (MTMS) and the organic homologue consists of poly(methyl methacrylate) (PMMA). The first part of the study offers new insights into systematically tuning preparation parameters leading to reproducible hydrophobic coating thicknesses that were highly robust and durable, regardless of the substrate nature. Treatment of SS with the two coatings under the same conditions confirmed the results found for GS. The last part of the study found that both MTMS-derived and PMMA-based coatings significantly reduced fetal bovine serum protein fouling with respect to uncoated substrates. The findings revealed that the dual-level coating morphology together with the molecular structure of MTMS and PMMA films are the key features in reducing protein fouling. The study is relevant for biomedical applications that require antifouling surfaces such as implant devices and surgical instruments.

Isolation and detection of exosomes via AAO membrane and QCM measurement

Increasing evidence suggests that exosomes are secreted by the majority of cancer cells and present in myriad biological fluids. They have been recognized as a potential new class of biomarkers for disease prognosis and diagnosis. The isolation and quantification of exosomes are crucial in facilitating exosome research. This paper presents a filtration method to isolate exosomes via anodic aluminum oxide (AAO) membrane and to quantify them using a quartz crystal microbalance (QCM). The filtration chip was designed to have two nano-porous AAO membranes sandwiched in between PDMS layers. In this study, fetal bovine serum (FBS) was adopted as the analyte for exosomal bioassays. The experimental results show that AAO membrane has a hydrophilic surface able to sieve exosomes by gravity. The spectrophotometric measurement and the result of gel electrophoresis confirmed that most of proteins in FBS were removed, leaving pure and undamaged exosomes for further analyses. Then, QCM measurement of the filtered FBS solution can give a quantitative analysis of exosome concentration. The relationship between the exosome concentration and its corresponding QCM's frequency shift is linear and monotonic. QCM is still sensitive to measure low concentration of exosomes.

Bright AIEgen–Protein Hybrid Nanocomposite for Deep and High‐Resolution In Vivo Two‐Photon Brain Imaging

AbstractTwo‐photon fluorescence imaging allows in vivo study of biological structures and activities in deep tissues, in which bright fluorophores with high photostability and good biocompatibility are highly desirable. Herein, a small‐molecule fluorogen with aggregation‐induced emission (AIEgen) is complexed with fetal bovine serum (FBS) proteins to develop a protein‐sized AIEgen–protein hybrid nanocomposite (TPEPy‐FBS) with bright far‐red/near‐infrared (NIR) emission, excellent photostability, and low phototoxicity for deep and high‐resolution in vivo two‐photon brain vasculature imaging. Upon complexation with FBS, the fluorescence of TPEPy is greatly intensified and a sixfold enhancement is observed with 10% FBS in aqueous media. The yielded TPEPy‐FBS shows good physical stability in aqueous media and the phototoxicity of TPEPy is dramatically inhibited after complexation with FBS. Moreover, TPEPy‐FBS exhibits bright two‐photon fluorescence in far‐red/NIR region and good photostability upon femtosecond laser excitation, which facilitates high performance in vivo imaging. A large imaging depth of 656 µm is obtained in brain vasculature network imaging with a high signal‐to‐background ratio of 234, where a small blood capillary of 1.05 µm can be resolved at an imaging depth of 656 µm. Highlighted is a simple and versatile strategy to develop efficient two‐photon probes for in vivo biological imaging.

Interaction of graphene oxide with cell culture medium: Evaluating the fetal bovine serum protein corona formation towards in vitro nanotoxicity assessment and nanobiointeractions

The interaction of single-layer graphene oxide (SLGO) and multi-layered graphene oxide (MLGO) with a cell culture medium (i.e. DMEM) was studied by evaluating fetal bovine serum (FBS) protein corona formation towards in vitro nanotoxicity assessment and nanobiointeractions. SLGO and MLGO exhibited different colloidal behavior in the culture medium, which was visualized by cryogenic transmission electron microscopy in situ analysis. Exploring proteomics and bioinformatics tools, 394 and 290 proteins were identified on the SLGO and MLGO hard corona compositions, respectively. From this amount, 115 proteins were exclusively detected on the SLGO and merely 11 on MLGO. SLGO enriched FBS proteins involved in metabolic processes and signal transduction, while MLGO enriched proteins involved in cellular development/structure, and lipid transport/metabolic processes. Such a distinct corona profile is due to differences on surface chemistry, aggregation behavior and the surface area of GO materials. Hydrophilic interactions were found to play a greater role in protein adsorption by MLGO than SLGO. Our results point out implications for in vitro studies of graphene oxide materials concerning the effective dose delivered to cells and corona bioactivity. Finally, we demonstrated the importance of integrating conventional and modern techniques thoroughly to understand the GO-FBS complexes towards more precise, reliable and advanced in vitro nanotoxicity assessment.