Combinatorial photoclickable peptide microarrays for high throughput screening of 3-D cellular microenvironments

Abstract

Event Abstract Back to Event Combinatorial photoclickable peptide microarrays for high throughput screening of 3-D cellular microenvironments Sadhana Sharma1, Michael Floren2, Wei Tan2 and Stephanie J. Bryant1 1 University of Colorado Boulder, Chemical and Biological Engineering, United States 2 University of Colorado Boulder, Mechanical Engineering, United States Statement of Purpose: Cell signaling, maturation, and differentiation processes are the outcome of a complex interplay between ligand-activated cell-matrix interactions and/or matrix physical properties such as elasticity and geometry[1],[2]. Microarrays have emerged as an important tool for a variety of applications including, but not limited to, study of disease biomarkers, immunomodulation, and cell processes, in a high-throughput manner[3]-[5]. Most of the existing ECM proteins/peptide arrays are either 2-D or shallow 3-D and, therefore, not able to capture the synergistic effects of all extrinsic and intrinsic cues on cell fate completely. Here, we report the development of a combinatorial peptide microarray platform for high throughput screening of 3-D cellular environments. Our system takes advantage of nanofibrous geometry, photoclickable thiol-ene poly(ethylene glycol) hydrogels, and orthogonality of thiol-ene polymerization that imparts good control over substrate elasticity, and enables postfunctionalization of the already prepared electrospun hydrogel substrates with ECM peptides with high reactivity and specificity. Methods: Four-arm poly(ethylene glycol) norbornene (PEGNB, 5 kDa, 10wt%), poly(ethylene glycol) dithiol (1 kDa), poly(ethylene oxide) (5 wt%, 400 kDa), and I2959 (0.1 wt%) were dissolved in DI water, electrospun (16 kV, 20 cm, 0.8 ml/hr), and collected as fibers (647.6 ± 81.4 nm) on a glass slide previously modified with 3-(mercaptopropyl) triethoxysilane. Substrates were subsequently exposed to UV (352 nm, 5 mW/cm2) light for specific time points to achieve elastic modulus ranging from 1-20 kPa. Microarrays were prepared using a 2470 Aushon arrayer (Fig 1A). Fluorescent dye (Alexa Fluor® 488/546 -C5 maleimide) or peptide (CRGDS, CRGES, YRGDS, CGDEAK-A568) in buffer (1% glycerol, 0.2% Triton X-100, 0.1% I2959) were printed and exposed to UV light. Cell studies were conducted using primary bovine pulmonary arterial smooth muscle cells (PASMCs). Following cell culture, substrates were fixed and subsequently stained with DAPI (cell nuclei) and A488-phallodin (F-actin cytoskeleton) to observe cell adhesion and spreading. Results: We have developed a peptide microarray platform based on electrospun photoclickable 3-D hydrogels for combinatorial screening of cellular microenvironments. Our system is capable of printing multiple biomolecules, simultaneously or sequentially, in different concentrations (in serial, random or custom design) as well-defined dots as illustrated using A488/546- maleimide dyes or cysteine-containing peptides (Fig 1B a-c; 1Cd-l). Fig 1C(g-l) demonstrates cell adhesion on CRGDS printed dots. Thiol group of Cys in CRGDS peptide enables its covalent conjugation to the substrates and thus promotes cell adhesion. Figure 1. A: Schematic of peptide printing process using Aushon Microarrayer. B: Combinatorial printing of A488/546- maleimide at several dilutions (serial and random). C: Representative confocal images of A488- maleimide or cells (PASMCs) cultured on CRGDS functionalized microarrays at several dilutions (serial and random) and custom spotting geometry (10 mM). Cell nuclear staining (DAPI, grey scale, g-i). DAPI (blue)/F-actin (green) (j-l). D: Selective cell adhesion and spreading on peptide printed microarrays. (m) CRGDS, (n) CRGES, (o) YRGDS, (p) CDGEAK-A568. Scale bars: 500 μm (a,b d,e,g,h,j,k), 2 mm (f,i,l), 100 μm (m-p). Fig 1D (m-p) demonstrates selective cell adhesion and spreading on peptide conjugated microarray. Cells strongly attach and spread on covalently conjugated CRGDS (m) but not to CRGES peptide (n). Only a few cells weakly attach to surface adsorbed YRGDS peptide (o). Printing of fluorescently labeled CDGEAK-A568 peptide establishes the covalent conjugation to our substrates (p). Our further studies also indicate that we can combinatorially print multiple peptides and screen cellular environments in a high throughput manner (data not shown). In addition, we can fine tune the elastic properties of our substrates to cover the entire range of elasticity relevant for cellular processes. Conclusions: We have developed a highly tunable combinatorial peptide microarray platform to facilitate high-throughput screening of engineered 3-D cellular microenvironments. NIH Grant # K25HL097246 and R01 HL119371; Mr. Markham and Linda Crnic Institute for Down Syndrome at University of Colorado Anschutz Medical Campus for use of the microarraying equipment