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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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Simplified schematicsof (A) cell adhesion to ECM protein FN viaintegrin interaction with FNIII9-10 cell-bindingdomains,cytoplasmic proteins, and the actin cytoskeleton to form a FAC and(B) a dynamic dual-ligand ECM model substrate. SAMs of alkyne-terminatedtetra(ethylene glycol) (alkyne-EG4SH) and tetra(ethyleneglycol)-terminated (EG4SH) alkanethiols are generated ongold substrates and reacted with hydroquinone- and azide-functionalizedcell adhesive peptide RGD (HQ-RGD-N3). HQ is considered“off” and can be turned “on” for oxyamine(OA) ligand conjugation by electrochemical oxidation. Ligands thatare functionalized with OA groups (i.e., peptides, KKKTTK-OA, PHRSN-OA,RGD-OA, cRGD-OA, and sugars, Man-OA, Gal-OA) react and conjugate toQ-presenting surfaces under physiological conditions. Cells are culturedand observed on surfaces displaying cell adhesive RGD and variablebiomolecule. In situ electrochemical reduction dynamically releasesthe variable biomolecule, and cellular response to this environmentalchange is monitored.
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fig1: Simplified schematicsof (A) cell adhesion to ECM protein FN viaintegrin interaction with FNIII9-10 cell-bindingdomains,cytoplasmic proteins, and the actin cytoskeleton to form a FAC and(B) a dynamic dual-ligand ECM model substrate. SAMs of alkyne-terminatedtetra(ethylene glycol) (alkyne-EG4SH) and tetra(ethyleneglycol)-terminated (EG4SH) alkanethiols are generated ongold substrates and reacted with hydroquinone- and azide-functionalizedcell adhesive peptide RGD (HQ-RGD-N3). HQ is considered“off” and can be turned “on” for oxyamine(OA) ligand conjugation by electrochemical oxidation. Ligands thatare functionalized with OA groups (i.e., peptides, KKKTTK-OA, PHRSN-OA,RGD-OA, cRGD-OA, and sugars, Man-OA, Gal-OA) react and conjugate toQ-presenting surfaces under physiological conditions. Cells are culturedand observed on surfaces displaying cell adhesive RGD and variablebiomolecule. In situ electrochemical reduction dynamically releasesthe variable biomolecule, and cellular response to this environmentalchange is monitored.

Mentions: Herein, we report a dynamic,redox-responsive strategy to immobilizeand release ligands in the presence of cells for cell adhesion, spreading,morphology, and migration studies (Figure 1). Electrochemistry enables the complete quantitative control overligand density and provides a dynamic molecular switch for the combinatorialdiscovery of ligand effects. Two bioothogonal coupling methodologies,click and oxime chemistry, were incorporated, and an HQ- and azide(N3)-functionalized RGD peptide (HQ-RGD) was synthesizedto modulate the ECM. Commercially available lysine-N3 andderivatized glycine-HQ were incorporated into Ser-Ser-Asp-Gly-Arg-Gly-C6 linker via solid-phase peptide synthesis to generate K(N3)-C6 linker-G(HQ)GRGDSS, where the N3 moiety is conjugated to alkyne-terminated SAMs via click chemistryand the HQ serves as a conjugation site for a variety of oxyamine-containingligands. Synergy peptide PHSRN, high affinity cyclic RGD (cRGD), putativeHS-binding sequence KKKTTK, and monosaccharides galactose (Gal) andmannose (Man) were functionalized with oxyamine groups and surveyedfor potential synergistic or antagonistic effects with cell-adhesiveRGD. Fibroblasts (Fbs) were seeded to the different ECM mimics substrates,with or without HQ-RGD, and the number of attached cells, spreadingarea, morphologies, and migration rates were tabulated. Inhibitionand competitive binding studies were also performed in which solubleFN, cRGD, and HS were added. Chondroitinase ABC and heparinase I andII were also delivered to Fbs in culture to determine whether HS-bindingKKKTTK exhibited a synergistic effect on cell adhesion and spreading.FAK protein levels were also detected and quantified. Furthermore,the ligands were released in the presence of cells, providing thedynamic component to our system, and cell adhesion, morphology, andmigration rates were again examined. To our knowledge, this is thefirst report that uses a density-controlled, bioorthogonal, and stimuli-responsivemodel ECM to probe ligand–cell surface integrin and syndecaninteractions in situ. The ability to switch ligands for the combinatorialscreening of synergistic of antagonistic ligand effects provides aplatform that would be of tremendous significance to the biosensorand biomaterials research communities.


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

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

Simplified schematicsof (A) cell adhesion to ECM protein FN viaintegrin interaction with FNIII9-10 cell-bindingdomains,cytoplasmic proteins, and the actin cytoskeleton to form a FAC and(B) a dynamic dual-ligand ECM model substrate. SAMs of alkyne-terminatedtetra(ethylene glycol) (alkyne-EG4SH) and tetra(ethyleneglycol)-terminated (EG4SH) alkanethiols are generated ongold substrates and reacted with hydroquinone- and azide-functionalizedcell adhesive peptide RGD (HQ-RGD-N3). HQ is considered“off” and can be turned “on” for oxyamine(OA) ligand conjugation by electrochemical oxidation. Ligands thatare functionalized with OA groups (i.e., peptides, KKKTTK-OA, PHRSN-OA,RGD-OA, cRGD-OA, and sugars, Man-OA, Gal-OA) react and conjugate toQ-presenting surfaces under physiological conditions. Cells are culturedand observed on surfaces displaying cell adhesive RGD and variablebiomolecule. In situ electrochemical reduction dynamically releasesthe variable biomolecule, and cellular response to this environmentalchange is monitored.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Simplified schematicsof (A) cell adhesion to ECM protein FN viaintegrin interaction with FNIII9-10 cell-bindingdomains,cytoplasmic proteins, and the actin cytoskeleton to form a FAC and(B) a dynamic dual-ligand ECM model substrate. SAMs of alkyne-terminatedtetra(ethylene glycol) (alkyne-EG4SH) and tetra(ethyleneglycol)-terminated (EG4SH) alkanethiols are generated ongold substrates and reacted with hydroquinone- and azide-functionalizedcell adhesive peptide RGD (HQ-RGD-N3). HQ is considered“off” and can be turned “on” for oxyamine(OA) ligand conjugation by electrochemical oxidation. Ligands thatare functionalized with OA groups (i.e., peptides, KKKTTK-OA, PHRSN-OA,RGD-OA, cRGD-OA, and sugars, Man-OA, Gal-OA) react and conjugate toQ-presenting surfaces under physiological conditions. Cells are culturedand observed on surfaces displaying cell adhesive RGD and variablebiomolecule. In situ electrochemical reduction dynamically releasesthe variable biomolecule, and cellular response to this environmentalchange is monitored.
Mentions: Herein, we report a dynamic,redox-responsive strategy to immobilizeand release ligands in the presence of cells for cell adhesion, spreading,morphology, and migration studies (Figure 1). Electrochemistry enables the complete quantitative control overligand density and provides a dynamic molecular switch for the combinatorialdiscovery of ligand effects. Two bioothogonal coupling methodologies,click and oxime chemistry, were incorporated, and an HQ- and azide(N3)-functionalized RGD peptide (HQ-RGD) was synthesizedto modulate the ECM. Commercially available lysine-N3 andderivatized glycine-HQ were incorporated into Ser-Ser-Asp-Gly-Arg-Gly-C6 linker via solid-phase peptide synthesis to generate K(N3)-C6 linker-G(HQ)GRGDSS, where the N3 moiety is conjugated to alkyne-terminated SAMs via click chemistryand the HQ serves as a conjugation site for a variety of oxyamine-containingligands. Synergy peptide PHSRN, high affinity cyclic RGD (cRGD), putativeHS-binding sequence KKKTTK, and monosaccharides galactose (Gal) andmannose (Man) were functionalized with oxyamine groups and surveyedfor potential synergistic or antagonistic effects with cell-adhesiveRGD. Fibroblasts (Fbs) were seeded to the different ECM mimics substrates,with or without HQ-RGD, and the number of attached cells, spreadingarea, morphologies, and migration rates were tabulated. Inhibitionand competitive binding studies were also performed in which solubleFN, cRGD, and HS were added. Chondroitinase ABC and heparinase I andII were also delivered to Fbs in culture to determine whether HS-bindingKKKTTK exhibited a synergistic effect on cell adhesion and spreading.FAK protein levels were also detected and quantified. Furthermore,the ligands were released in the presence of cells, providing thedynamic component to our system, and cell adhesion, morphology, andmigration rates were again examined. To our knowledge, this is thefirst report that uses a density-controlled, bioorthogonal, and stimuli-responsivemodel ECM to probe ligand–cell surface integrin and syndecaninteractions in situ. The ability to switch ligands for the combinatorialscreening of synergistic of antagonistic ligand effects provides aplatform that would be of tremendous significance to the biosensorand biomaterials research communities.

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