Problem of Diminished cRGD Surface Activity and What Can Be Done about It

Abstract

RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from in vitro experiments probing fundamental molecular mechanisms of cell adhesion to more applied in vivo strategies in medical imaging and cancer therapeutics. To better understand the outcomes of RGD-based approaches, we quantified the degree to which cyclic RGD (cRGD) activity is blocked by nonspecific binding of commonly used medium constituents. First, we show that recombinant αVβ3 integrins can be used as a highly sensitive cell-free sensor to quantitatively and reliably characterize the activity of cRGD-functionalized surfaces via surface plasmon resonance (SPR). Next, SPR experiments were utilized to measure the extent of blocking of cRGD-functionalized surfaces by the commonly used agents BSA, PLL-g-PEG, and fetal calf serum (FCS)-supplemented media, using recombinant αVβ3 integrin as a probe for cRGD binding activity in the presence of blocking agents. All three additives were highly efficient blockers of cRGD activity, as exemplified by cell culture media containing 1% FCS which reduced the cRGD activity by 33-fold. We then developed a strategy to combat these deleterious effects by employing the recombinant integrins as a protective cap. We show that the unblocked cRGD activity can be preserved in the presence of PLL-g-PEG by employing the αVβ3 integrin as a removable protective cap, both in cell-free and in vitro experiments. In vitro studies with MDA-MB-231 cells cultured atop cRGD-functionalized surfaces found that cell adhesion and migration prevented by PLL-g-PEG were restored when this protective cap approach was used.

A Self-Supervised Machine Learning Approach for Objective Live Cell Segmentation and Analysis

“…Fig 4a-c shows the tracking of several MDA-MB-231 cells segmented via the self-supervised approach under 10X phase contrast microscopy on cRGD functionalized gold coverslips. 31 Fig 4a shows the labeled tracks of the cells’ centroids over the course of 400 minutes, with the corresponding initial and final image shown in Figs 4b,c. The cell associated with track 2 undergoes mitosis at approximately 320 minutes, creating two new tracks (5 and 6) for the daughter cells.…”

Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity

“…The surfaces and thiol design specifications defined the roughness, stiffness and tether length parameter space of the experiments: the surfaces are ultra smooth with an average surface roughness of 0.5 nm based on AFM characterization 30 ; the substrate stiffness is maximal due to direct conjugation of the cRGD peptide to the underlying Au/glass substrate (> 50 GPa versus ~ 1kPa -1MPa for most hydrogel constructs); the tether length, based on the constituent alkane and PEG linker lengths, is 3.8 nm which is categorized as short for PEG tether linker length studies that typically run from several nanometers to hundreds on nanometers 19,32 . As will be discussed in detail below, the surface activity was systematically varied by adjustments of the SPO:SPC ratio.…”

“…We emphasize the word active in this work because we have previously shown that RGD surface activity can be dramatically reduced by blocking from a variety of common media and surface functionalization components (e.g. serum, BSA and PLL-PEG), which in turn affects cell phenotype 30 . Thus, even when using sophisticated lithographic approaches, the active ligand density can vary with laboratory methodology.…”

“…Live cell experiments are conducted on the in-parallel produced in vitro chip -an approach analogous to other in vitro surface characterization workflows such as those used for surface roughness or stiffness. The technique described here has multiple advantages: (1) receptor-ligand kinetics as characterized by SPR has been demonstrated to be reproducible across SPR platforms and laboratories, thereby enabling the standardization of surface activity measurements 31 ; (2) we show the SPR technique has the sensitivity to measure ligand activity across the range of surface densities necessary to trigger cell adhesion and commonly encountered in the extracellular environment; (3) the SPR chip offers a straightforward means of not only characterizing ligand activity but also whether culture media or other added factors degrade ligand activity, and if so, by how much 30 ; (4) we demonstrate that when ligand spacing studies are investigated using both SPR and in vitro approaches, a comparison of the results can provide biological insights into the nature of cellular adhesion and dynamics. For SPR measurements, commercially available bare gold sensor chips were purchased from Cytiva (SIA Kit Au).…”

“…For in vitro studies, 20 nm Au thin films with sub-nm surface roughness were deposited on 25.4 mm diameter, No. 1.5 glass coverslips by e-beam evaporation as previously described 30 . The gold film was thin enough to allow for phase contrast microscopy techniques to be employed but thick enough for a continuous film to be formed on the substrate.…”