4 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
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
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
Distribution Profiling of Circulating MicroRNAs in Serum
Circulating microRNAs (miRNAs) are
potential biomarkers useful
in cancer diagnosis. They have been found to be bound to various carriers
like proteins, lipoprotein particles, and exosomes. It is likely that
only miRNAs in particular carriers, but not the overall quantity,
are directly related to cancer development. Herein, we developed a
method for rapid separation of different miRNA carriers in serum using
asymmetrical flow field flow fractionation (AF4). Sera from two healthy
individuals (control) or from two cancer patients (case) were fractionated.
Six fractions enriching different types of miRNA carriers, such as
the lipoprotein particles and exosomes, were collected. The quantities
of eight selected miRNAs in each fraction were obtained by RT-qPCR
to yield their distribution profiles among the carriers. Larger changes
in miRNA quantity between the control and the case were detected in
the fractionated results compared to the sum values. Statistical analysis
on the distribution profiles also proved that, the quantities of 4
miRNAs within particular fractions showed significant difference between
the controls and the cases. On the contrary, if the overall quantity
of the miRNA was subject to the same statistical analysis, only 2
miRNAs exhibited significant difference. Moreover, principle component
analysis revealed good separation between the controls and the cases
with the fractionated miRNA amounts. All in all, we have demonstrated
that, our method enables comprehensive screening of the distribution
of circulating miRNAs in the carriers. The obtained distribution profile
enlarges the miRNA expression difference between healthy individuals
and cancer patients, facilitating the discovery of specific miRNA
biomarkers for cancer diagnosis