18 research outputs found
Size and Surface Functionalization of Iron Oxide Nanoparticles Influence the Composition and Dynamic Nature of Their Protein Corona
Nanoparticles (NPs) adsorb proteins
when in the biological matrix, and the resulted protein corona could
affect NP-cell interactions. The corona has a dynamic nature with
the adsorbed proteins constantly exchanging with the free proteins
in the matrix at various rates. The rapidly exchanging proteins compose
the soft corona, which responds more dynamically to environment changes
than the hard corona established by the ones with slow exchange rates.
In the present study, the corona formed on the superparamagnetic iron
oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation
and ultracentrifugation, which rapidly differentiated the corona proteins
based on their exchange rates. By varying the surface hydrophobicity
of the SPIONs with a core size around 10 nm, we found out that, the
more hydrophobic surface ligand attracted proteins with higher surface
hydrophobicity and formed a more dynamic corona with a larger portion
of the involved proteins with fast exchange rates. Increasing the
core diameter of the SPIONs but keeping the surface ligand the same
could also result in a more dynamic corona. A brief investigation
of the effect on the cellular uptake of SPIONs using one selected
corona protein, transferrin, was conducted. The result showed that,
only the stably bound transferrin could significantly enhance cellular
uptake, while transferrin bound in a dynamic nature had negligible
impact. Our study has led to a better understanding of the relationship
between the particle properties and the dynamic nature of the corona,
which can help with design of nanomaterials with higher biocompatibility
and higher efficacy in biosystems for biomedical applications
ZrO<sub>2</sub> Nanofiber as a Versatile Tool for Protein Analysis
Phosphorylation
is one of the most important post-translational
modifications in proteins. Their essential roles in the regulation
of cellular processes and alteration of protein–protein interaction
networks have been actively studied. However, phosphorylated proteins
are present at low abundance in cells, and ionization of the modified
peptides is often suppressed by the more abundant species in mass
spectrometry. Effective enrichment techniques are needed to remove
the unmodified peptides and concentrate the phosphorylated ones before
their identification and quantification. Herein, we prepared ZrO<sub>2</sub> nanofibers by electrospinning, a straightforward and easy
fabrication technique, and applied them to enrich phosphorylated peptides
and proteins. The fibers showed good size homogeneity and porosity
and could specifically bind to the phosphorylated peptides and proteins,
allowing their separation from the unmodified analogues when present
in either simple protein digests or highly complex cell lysates. The
enrichment performance was superior to that of the commercially available
nanoparticles. Moreover, modifying the solution pH could lead to selective
adsorption of proteins with different p<i>I</i> values,
suggesting the fibers’ potential applicability in charge-based
protein fractionation. Our results support that the electrospun ZrO<sub>2</sub> nanofibers can serve as a versatile tool for protein analysis
with great ease in preparation and handling
Low-Dose Exposure of WS<sub>2</sub> Nanosheets Induces Differential Apoptosis in Lung Epithelial Cells
Escalating the production and application of tungsten
disulfide
(WS2) nanosheets inevitably increases environmental human
exposure and warrants the necessity of studies to elucidate their
biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 μg/mL)
on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells.
The low doses, which approximate real-world exposures, were found
to induce cell apoptosis, while doses ≥ 50 μg/mL cause
necrosis. Focused studies on low-dose exposure to WS2 nanosheets
revealed more details of the impacts on both cell lines, including
reduction of cell metabolic activity, induction of lipid peroxidation
in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation
that led to the loss of ATP production. These phenomena, along with
the expression situations of a few key proteins involved in apoptosis,
point toward the occurrence of mitochondria-dependent apoptotic signaling
in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were
noticed as well. Specifically, BEAS-2B cells experienced more adverse
effects and took up more nanosheets than A549 cells. Our results highlight
the importance of dose and cell model selection in the assessment
of nanotoxicity. By using doses consistent with real-world exposures
and comparing normal and diseased cells, we can gain knowledge to
guide the development of safety precautions for mitigating the adverse
impacts of nanomaterial exposure on human health
Detection of Femtomolar Proteins by Nonfluorescent ZnS Nanocrystal Clusters
Cation exchange (CX) in the nonfluorescent ZnS nanocrystal
clusters
(NCCs) was employed to detect trace biomolecules with immunoassays.
The NCCs were porous and allowed fast cation exchange reaction to
release an ultralarge number of Zn<sup>2+</sup> from each cluster
that turned on the Zn-responsive dyes for fluorescence detection.
The ZnS NCCs were highly stable in biological buffers and more biocompatible
than quantum dots. Zn<sup>2+</sup> release efficiency and target binding
by NCCs with average diameters of 44 nm, 86 nm, and 144 nm were investigated.
The smallest NCCs exhibited the highest CX efficiency because of its
larger surface area and bigger pores inside the cluster structure,
and 71.0% of the enclosed Zn<sup>2+</sup> were freed by CX with 2-min
microwave irradiation. They also experienced the least space hindrance
and the fastest rate when binding to target molecules immobilized
on surface. When the 44-nm NCCs were used to detect IgE in a sandwich
assay, the limit of detection (LOD) was 5 pg/mL (33 fM), 1,000 times
better than that of ELISA. Our results well demonstrate that CX in
the ZnS NCCs is superior to the conventional signaling strategies
in its high amplification efficiency, robustness, and biocompatibility
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Fluorescamine Labeling for Assessment of Protein Conformational Change and Binding Affinity in Protein–Nanoparticle Interaction
Protein
adsorption alters the “biological identity”
of nanoparticles (NPs) and could affect how biosystems respond to
invading NPs. Study of protein–NP interaction can help understand
how the physicochemical properties of NPs impact the interaction and
thus potentially guide the design of safer and more effective NPs
for biomedical or other applications. Binding affinity between proteins
and NPs and the occurrence of protein conformational change upon binding
to NPs are two important aspects to be learned, but few methods are
currently available to assess both simultaneously in a simple way.
Herein, we demonstrated that the fluorescamine labeling method developed
by our group not only could reveal protein conformational change upon
adsorption to NPs, owing to its capability to label the primary amines
exposed on protein surface, but also could be applied to measure the
binding affinity. By screening the interaction between a large number
of proteins and four types of NPs, the present study also revealed
that protein adsorption onto NPs could be strongly affected by structure
flexibility. The proteins with high structure flexibility experienced
high degrees of conformation change when binding to the polystyrene
NPs, which could potentially influence protein function. Overall,
we demonstrate that our assay is a quick, simple, and high-throughput
tool to reveal potential impacts on protein activity and evaluate
the strength of protein–NP binding
Exponential Strand-Displacement Amplification for Detection of MicroRNAs
MicroRNAs
(miRNAs) are promising targets for disease diagnosis. However, miRNA
detection requires rapid, sensitive, and selective detection to be
effective as a diagnostic tool. Herein, a miRNA-initiated exponential
strand-displacement amplification (SDA) assay was reported. With the
Klenow fragment, nicking enzyme <i>Nt.AlwI</i>, and two
primers, the miRNA target can trigger two cycles of nicking, polymerization,
and displacement reactions. These reaction cycles amplified the target
miRNA exponentially and generated dsDNAs detectable with SYBR Green
I in real-time PCR. As low as 16 zmol of the target miRNA was detected
by this one-pot assay within 90 min, and the dynamic range spanned
over 9 orders of magnitude. Negligible impact from the complex biological
matrix was observed on the amplification reaction, indicating the
assay’s capability to directly detect miRNAs in biofluids
Dissociation-Based Screening of Nanoparticle–Protein Interaction via Flow Field-Flow Fractionation
A protein
corona will be formed on nanoparticles (NPs) entering a biological
matrix, which can influence particles’ subsequent behaviors
inside the biological systems. For proteins bound stably to the NPs,
they can exhibit different association/dissociation rates. The binding
kinetics could affect interaction of the NPs with cell surface receptors
and possibly contribute to the outcomes of NPs uptake. In the present
study, a method to differentiate the corona proteins based on their
relative dissociation rates from the NPs was developed, employing
flow field-flow fraction (F4) in combination with centrifugation.
The proteins bound to the superparamagnetic iron oxide NPs (SPION)
present in an IgG/albumin depleted serum were isolated via collection
of the SPIONs by either F4 or centrifugation. They were subsequently
analyzed by LC-MS/MS and identified. Because the SPION-protein complexes
injected to F4 dissociated continuously under the nonequilibrium separation
condition, only the proteins with slow enough dissociation rates would
be collected with the NPs in the eluent of F4. However, in centrifugation,
proteins with good affinity to the SPIONs were collected regardless
of the dissociation rates of the complexes. In both cases, the nonbinding
ones were washed off. Capillary electrophoresis and circular dichroism
were employed to verify the binding situations of a few SPION-protein
interactions, confirming the effectiveness of our method. Our results
support that our method can screen for proteins binding to NPs with
fast on-and-off rates, which should be the ones quickly exchanging
with the free matrix proteins when the NPs are exposed to a new biological
media. Thus, our method will be useful for investigation of the temporal
profile of protein corona and its evolution in biological matrices
as well as for high-throughput analysis of the dynamic feature of
protein corona related to particle properties
Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation
Extracellular vesicles (EVs) play important roles in
cell–cell
communication and pathological development. Cargo profiling for the
EVs present in clinical specimens can provide valuable insights into
their functions and help discover effective EV-based markers for diagnostic
and therapeutic purposes. However, the highly abundant and complex
matrix components pose significant challenges for specific identification
of low-abundance EV cargos. Herein, we combine asymmetrical flow
field-flow fractionation (AF4) with large-volume sample stacking and
capillary electrophoresis (LVSS/CE), to attain EVs with high purity
for downstream protein profiling. This hyphenated system first separates
the EVs from the contamination of smaller serum proteins by AF4,
and second resolves the EVs from the coeluted, nonvesicular matrix
components by CE following LVSS. The optimal LVSS condition permits
the injection of 10-fold more EVs into CE compared to the nonstacking
condition without compromising separation resolution. Collection and
downstream analysis of the highly pure EVs after CE separation were
demonstrated in the present work. The high EV purity yields a much-improved
labeling efficiency when detected by fluorescent antibodies compared
to those collected from the one-dimension separation of AF4, and permits
the identification of more EV-specific cargos by LC–MS/MS compared
to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit,
and AF4. Our results strongly support that AF4-LVSS/CE can improve
EV isolation and cargo analysis, opening up new opportunities for
understanding EV functions and their applications in the biomedical
fields
High-Throughput Profiling of Nanoparticle–Protein Interactions by Fluorescamine Labeling
A rapid, high throughput fluorescence
assay was designed to screen
interactions between proteins and nanoparticles. The assay employs
fluorescamine, a primary-amine specific fluorogenic dye, to label
proteins. Because fluorescamine could specifically target the surface
amines on proteins, a conformational change of the protein upon interaction
with nanoparticles will result in a change in fluorescence. In the
present study, the assay was applied to test the interactions between
a selection of proteins and nanoparticles made of polystyrene, silica,
or iron oxide. The particles were also different in their hydrodynamic
diameter, synthesis procedure, or surface modification. Significant
labeling differences were detected when the same protein incubated
with different particles. Principal component analysis (PCA) on the
collected fluorescence profiles revealed clear grouping effects of
the particles based on their properties. The results prove that fluorescamine
labeling is capable of detecting protein–nanoparticle interactions,
and the resulting fluorescence profile is sensitive to differences
in nanoparticle’s physical properties. The assay can be carried
out in a high-throughput manner, and is rapid with low operation cost.
Thus, it is well suited for evaluating interactions between a larger
number of proteins and nanoparticles. Such assessment can help to
improve our understanding on the molecular basis that governs the
biological behaviors of nanomaterials. It will also be useful for
initial examination of the bioactivity and reproducibility of nanomaterials
employed in biomedical fields
Separation of Methylated Histone Peptides via Host-Assisted Capillary Electrophoresis
Lysine
methylation in protein is one important epigenetic mechanism
that regulates diverse biological processes but is challenging to
study due to the large variability in methylation levels and sites.
Here, we show that supramolecular hosts such as calixarenes and cucurbiturils
can be applied in the background electrolyte (BGE) of capillary electrophoresis
(CE) for highly effective separation of post-translationally methylated
histone peptides. The molecular recognition event causes a shift in
the electrophoretic mobility of the peptide, allowing affinity measurement
for binding between the synthetic receptor and various methylated
lysine species. Successful separation of the H3 peptides carrying
different methylation levels at the K9 position can be achieved using <b>CX4</b> and <b>CX6</b> as the BGE additives in CE, enabling
monitoring of the activity of the histone lysine demethylase JMJD2E.
This reveals the power of combining high resolution CE with synthetic
hosts for study of protein methylation, and the method should be capable
of analyzing complex biological samples for better understanding of
the functions of histone methylation