Authors
Beatrix Peter, Eniko Farkas, Eniko Forgacs, Andras Saftics, Boglarka Kovacs, Sandor Kurunczi,
Inna Szekacs, Antal Csampai, Szilvia Bosze, and Robert Horvath
Abstract
The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ
monitored.
After, the kinetics of cellular adhesion on the EGCg exposed
coatings were recorded in real-time.
The employed plate-based waveguide
biosensor is applicable to monitor small molecule binding and sensitive
to sub-nanometer scale changes in cell membrane position and cell mass
distribution; while detecting the signals of thousands of adhering
cells.
The combination of this remarkable sensitivity and throughput
opens up new avenues in testing complicated models of cell-surface
interactions.
The systematic studies revealed that, despite the reported
excellent antifouling properties of the coatings, EGCg strongly
interacted with them, and affected their cell adhesivity in a
concentration dependent manner.
Moreover, the differences between the
effects of the fresh and oxidized EGCg solutions were first
demonstrated.
Using a semiempirical quantumchemical method we showed
that EGCg binds to the PEG chains of PLL-g-PEG-RGD and
effectively blocks the RGD sites by hydrogen bonds.
The calculations
supported the experimental finding that the binding is stronger for the
oxidative products.
Our work lead to a new model of polyphenol action on
cell adhesion ligand accessibility and matrix rigidity.
Detection
of cellular adhesion is of outstanding diagnostic and basic research
utility.
On the one hand, changes in cell adhesivity can be a sign for
various diseases; e.g. the variety of integrins, a major family of cell
adhesion receptors that bind to the extracellular matrix (ECM), changes
during tumor transformation.
On the other hand, measurement of the effect of bioactive substances on
the adhesion of cancer cells can be an effective tool in the design of
antineoplastic pharmaceuticals.
By controlling interactions between a
cell and its ECM, cell behavior and function can be influenced.
Under in vivo
conditions, the cell adhesion involves several components, these are
interacting in a complicated and tightly controlled manner, still under
intense research.
These components are the proteins and carbohydrates of
the extracellular matrix, the cell adhesion receptors and other soluble
factors (ions, small molecules) regulating the interactions.
In
contrast, due to experimental difficulties, most experimental models
resulting in quantitative data about the cellular adhesion can be
considered as a strong simplification of the in vivo situation.
A wide range of experimental methods are available to measure cell adhesion and cell–surface interactions.
However, most of them have serious disadvantages when a multicomponent
model of cell adhesion has to be quantitatively investigated in a
reasonable time frame.
For example, labeling techniques use fluorescent
markers that may affect normal cell behavior and the imaging time is
often limited by the bleaching of the marker.
Furthermore, dyes may
interact with the sample material itself.
Some techniques usually
involve complicated and time-consuming steps and are not available in
high-throughput format.
Consequently, it is difficult to do large number
of parallel measurements simultaneously, and sometimes it can easily
take months to execute all of the experiments required.
These techniques are especially promising when the kinetics of
interactions have to be investigated. Sensitivity and detection capacity
used to be considered as obstacles of the widespread use of label-free
detection, but recent developments have by far overcome these limitations.
While quartz crystal microbalance (QCM), cellular dielectric spectroscopy (CDS), optical waveguide lightmode spectroscopy (OWLS), surface plasmon resonance (SPR)
usually employ one or a low number of sensing units, novel biosensors
have high-throughput capability to practically parallel measurements of
hundreds of samples in a microplate format.
At present, they easily meet
the required sensitivity of being able to detect the binding of ligands
of molecular mass as low as 100–200 Da, below 5 pg/mm2
surface mass density; and their current throughput allows up to 460,000
data points/hour. These include electric cell–substrate impedance
sensing (ECIS), photonic crystal based sensors, and resonance waveguide grating (RWG).
Moreover, it has been proven that optical waveguide based sensors are
capable of investigating not just biological samples, but nanoparticles
and self-assembled nanostructured coatings as well.
Tea polyphenol is a mixture of compounds such as flavanoids and tannins found in green tea. Tea polyphenol acts as an antioxidant, and is beneficial for coronary artery disease treatment and cancer reduction. Tea polyphenol
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