The Problem of Diminished cRGD Surface Activity and What Can Be Done About It.
2020
RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from in vitro experiments prob-ing 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 non-specific binding of com-monly used media constituents. First, we show that recombinant Vbeta3 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 blocking of cRGD functionalized surfaces by the commonly used agents BSA, PLL-g-PEG and fetal calf serum (FCS) supplemented media, using recombinant Vbeta3 integrin as a probe of 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 cRGD activity 33 fold. We then developed a strategy to combat these deleterious effects by employing the recombi-nant 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 Vbeta3 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 functionalize surfaces found that cell adhesion and migration prevented by PLL-g-PEG was restored when this protective cap approach was used.
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