Differential Modulation of Biological Properties In Vitro by a Range of cRGDY Peptides on Clinically Translated Dual-Modality Silica Nanoparticles

Introduction: Ultra-small Dual Modality nanoparticles, termed Cornell dots (i.e., C’ dots) may provide a disease-directed platform for delivery of small cancer molecules for theranostic purposes. For clinical advancement of... [ view full abstract ]

Introduction:
Ultra-small Dual Modality nanoparticles, termed Cornell dots (i.e., C’ dots) may provide a disease-directed platform for delivery of small cancer molecules for theranostic purposes. For clinical advancement of such platforms is the need to characterize at the cellular and molecular levels, to light biological properties of these probes. We used silica nanoparticle encapsulate Cy 5 dyes (C’ dots), surface functionalized with methoxy-terminated polyethylene glycol (PEG), that bind to it different numbers of integrin-targeting cRGDY ligands (N-6, 14, 18) (i.e., cRGDY-PEG-C’ dots). Their activities were compared in vitro on several biological systems using cells that over express the αvβ3 receptor, a human melanoma (M21) and human umbilical vein endothelial (HUVEC) cells.
Methods :
Binding and internalization - flow cytometry; Migration performed by time lapse. Adhesion and spreading - SpectraMax5 micro plate reader; Vi-Cell Counter for cell Viability, Effect on signaling - Western blot analysis.
Results:
Dose response and time-dependent binding revealed binding saturation at 50 nM with 14 or 18 cRGDY-PEG-C’ dots at two hours as opposed to 100 nM with 6 cRGDY-PEG-C’ dots at four hours incubation. Internalization studies revealed a higher uptake (15-20%) of the 14 and 18 particles in 370C compared to 6 cRGDY-PEG-C’ dots on M21. Binding specificity was demonstrated using antibodies against the αvβ3 receptor. There was no binding of any of the particle on cells that don’t express the αv receptor subunit. Minor binding was observed with two different sizes of scramble peptide-cRADY (6 or 15-20). Additionally, a higher migration rate of HUVEC cells, adhesion with spreading on fibronectin coated plates of M21 cells and proliferation of M21 cells were shown with 14 or 18 cRGDY-PEG-C’ dots compared to 6 cRGDY-PEG-C’ dots. Activation of the FAK pathway by integrin was also several fold (x2) more with 14 cRGDY as opposed to 6 cRGDY on the particle.
Discussion:
Changes in vitro on the level of binding and key biological processes-cell migration, adhesion/spreading, proliferation process were greater with 14-18 cRGDY-PEG-C’ dots. This paves the way in designing a diagnostic platform for primary tumor and cancer metastasis or therapeutic purpose for processes like angiogenesis and wound healing.