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In situ modulation of cell behavior via smart dual-ligand surfaces.

Pulsipher A, Park S, Dutta D, Luo W, Yousaf MN - Langmuir (2014)

Bottom Line: Such tools provide strategies for identifying specific ligand-receptor interactions that induce vital biological consequences.A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared.The identity and nature of the dual-ligand combination directly influenced cell behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.

ABSTRACT
Due to the highly complex nature of the extracellular matrix (ECM), the design and implementation of dynamic, stimuli-responsive surfaces that present well-defined ligands and serve as model ECM substrates have been of tremendous interest to biomaterials, biosensor, and cell biology communities. Such tools provide strategies for identifying specific ligand-receptor interactions that induce vital biological consequences. Herein, we report a novel dual-ligand-presenting surface methodology that modulates dynamic ECM properties to investigate various cell behaviors. Peptides PHSRN, cRGD, and KKKTTK, which mimic the cell- and heparan sulfate-binding domains of fibronectin, and carbohydrates Gal and Man were combined with cell adhesive RGD to survey possible synergistic or antagonist ligand effects on cell adhesion, spreading, growth, and migration. Soluble molecule and enzymatic inhibition assays were also performed, and the levels of focal adhesion kinase in cells subjected to different ligand combinations were quantified. A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared. The identity and nature of the dual-ligand combination directly influenced cell behavior.

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Number of adhered cellsafter 2 h of culture on the following substrates:single ligands (green) cRGD, Man, KKKTTK, Gal, or PHSRN; dual ligands(blue) HQ-RGD + cRGD, Man, KKKTTK, Gal, or PHSRN. Statistical analyseswere performed with respect to HQ-RGD: *p < 0.001and **p < 0.01.
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fig2: Number of adhered cellsafter 2 h of culture on the following substrates:single ligands (green) cRGD, Man, KKKTTK, Gal, or PHSRN; dual ligands(blue) HQ-RGD + cRGD, Man, KKKTTK, Gal, or PHSRN. Statistical analyseswere performed with respect to HQ-RGD: *p < 0.001and **p < 0.01.

Mentions: Before testing our dual-ligandECM system, we first verified whether Fbs could adhere and healthilyspread on substrates presenting HQ-RGD and each decoupled ligand (Figure 2). As such, HQ-RGD and cRGD, Man, Gal, PHSRN, andKKKTTK ligands were immobilized to alkyne-EG4SH (20 mMin H2O/EtOH (3:1), cat CuSO4·5H2O and NaAsc, 90 min) and HQ-terminated (20 mM in PBS, 2 h) SAMs,respectively, at a 2% ligand density. Fbs cells were then seeded tosurfaces (∼103 cells/mL, 2 h), fixed, imaged, andcounted after 2 h. As shown in Figure 2, allligands were able to support cell adhesion to varying degrees. Thedata are represented as the average number of attached cells per 4×frame (eight random regions). HQ-RGD and cRGD demonstrated similaraffinity for attracting Fbs when compared to the surfaces presentingMan, Gal, KKKTTK, and PHSRN, which exhibited almost a 2-fold reductionin the amount of attached Fbs. Additionally, the Fbs were more looselyadhered and adopted a rounder morphology on substrates presentingMan, Gal, KKKTTK, and PHSRN. However, when these ligands were combinedwith and immobilized to SAMs with 1% Q-RGD (2% ligand total), theamount of cells increased significantly, approximately 2-fold, resemblingthe results observed with 2% HQ-RGD. When Man and Gal were coupledto HQ-RGD, Fbs behaved similarly to when in the presence of cell adhesiveRGD; similar numbers of attached cells and mor phologies were observed.When PHSRN was coupled to HQ-RGD, a synergistic effect on the amountof cells adhered and over cell spreading and morphology was observed,although, at surface value, the actual number of attached cells didnot increase significantly from the substrates with 2% HQ-RGD. Surprisingly,KKKTTK coupled to HQ-RGD showed a dramatic increase in the amountof attached Fbs when compared to HQ-RGD- and KKKTTK-presenting substrates,indicating a profound synergistic effect between KKKTTK and RGD onadhesion. A marked 0.5-fold increase from HQ-RGD substrates was observedon KKKTTK + HQ-RGD.


In situ modulation of cell behavior via smart dual-ligand surfaces.

Pulsipher A, Park S, Dutta D, Luo W, Yousaf MN - Langmuir (2014)

Number of adhered cellsafter 2 h of culture on the following substrates:single ligands (green) cRGD, Man, KKKTTK, Gal, or PHSRN; dual ligands(blue) HQ-RGD + cRGD, Man, KKKTTK, Gal, or PHSRN. Statistical analyseswere performed with respect to HQ-RGD: *p < 0.001and **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4334223&req=5

fig2: Number of adhered cellsafter 2 h of culture on the following substrates:single ligands (green) cRGD, Man, KKKTTK, Gal, or PHSRN; dual ligands(blue) HQ-RGD + cRGD, Man, KKKTTK, Gal, or PHSRN. Statistical analyseswere performed with respect to HQ-RGD: *p < 0.001and **p < 0.01.
Mentions: Before testing our dual-ligandECM system, we first verified whether Fbs could adhere and healthilyspread on substrates presenting HQ-RGD and each decoupled ligand (Figure 2). As such, HQ-RGD and cRGD, Man, Gal, PHSRN, andKKKTTK ligands were immobilized to alkyne-EG4SH (20 mMin H2O/EtOH (3:1), cat CuSO4·5H2O and NaAsc, 90 min) and HQ-terminated (20 mM in PBS, 2 h) SAMs,respectively, at a 2% ligand density. Fbs cells were then seeded tosurfaces (∼103 cells/mL, 2 h), fixed, imaged, andcounted after 2 h. As shown in Figure 2, allligands were able to support cell adhesion to varying degrees. Thedata are represented as the average number of attached cells per 4×frame (eight random regions). HQ-RGD and cRGD demonstrated similaraffinity for attracting Fbs when compared to the surfaces presentingMan, Gal, KKKTTK, and PHSRN, which exhibited almost a 2-fold reductionin the amount of attached Fbs. Additionally, the Fbs were more looselyadhered and adopted a rounder morphology on substrates presentingMan, Gal, KKKTTK, and PHSRN. However, when these ligands were combinedwith and immobilized to SAMs with 1% Q-RGD (2% ligand total), theamount of cells increased significantly, approximately 2-fold, resemblingthe results observed with 2% HQ-RGD. When Man and Gal were coupledto HQ-RGD, Fbs behaved similarly to when in the presence of cell adhesiveRGD; similar numbers of attached cells and mor phologies were observed.When PHSRN was coupled to HQ-RGD, a synergistic effect on the amountof cells adhered and over cell spreading and morphology was observed,although, at surface value, the actual number of attached cells didnot increase significantly from the substrates with 2% HQ-RGD. Surprisingly,KKKTTK coupled to HQ-RGD showed a dramatic increase in the amountof attached Fbs when compared to HQ-RGD- and KKKTTK-presenting substrates,indicating a profound synergistic effect between KKKTTK and RGD onadhesion. A marked 0.5-fold increase from HQ-RGD substrates was observedon KKKTTK + HQ-RGD.

Bottom Line: Such tools provide strategies for identifying specific ligand-receptor interactions that induce vital biological consequences.A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared.The identity and nature of the dual-ligand combination directly influenced cell behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.

ABSTRACT
Due to the highly complex nature of the extracellular matrix (ECM), the design and implementation of dynamic, stimuli-responsive surfaces that present well-defined ligands and serve as model ECM substrates have been of tremendous interest to biomaterials, biosensor, and cell biology communities. Such tools provide strategies for identifying specific ligand-receptor interactions that induce vital biological consequences. Herein, we report a novel dual-ligand-presenting surface methodology that modulates dynamic ECM properties to investigate various cell behaviors. Peptides PHSRN, cRGD, and KKKTTK, which mimic the cell- and heparan sulfate-binding domains of fibronectin, and carbohydrates Gal and Man were combined with cell adhesive RGD to survey possible synergistic or antagonist ligand effects on cell adhesion, spreading, growth, and migration. Soluble molecule and enzymatic inhibition assays were also performed, and the levels of focal adhesion kinase in cells subjected to different ligand combinations were quantified. A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared. The identity and nature of the dual-ligand combination directly influenced cell behavior.

Show MeSH
Related in: MedlinePlus