Heparin-functionalized thermoresponsive surface

Abstract

Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.

Keywords: :

• poly(N-isopropylacrylamide)
• 2-carboxyisopropylacrylamide
• thermoresponsive cell culture surface
• heparin
• basic fibroblast growth factor
• cell sheet

This article refers to:

Interactions between bioactive ligands and membrane receptors on the cellular surface play essential roles in regulating cellular adhesion, proliferation, migration, differentiation, and signal transduction. Extracellular matrix (ECM) such as fibronectin and collagen provides a microenvironment to mediate the interactions between bioactive ligands and their cell membrane receptors. Typically, a peptide sequence Arg-Gly-Asp (RGD) found in fibronectin, type I collagen, and other ECMs is a common element in cellular recognition for integrin-mediated cell adhesion.¹ These synthetic ligands that are immobilized onto cell culture surfaces serve as a useful tool to stimulate cell adhesion on various materials.²

The use of stimuli-responsive materials such as thermoresponsive polymers for switching sequences facilitates the dynamic regulation of the interactions between bioactive ligands and their cell membrane receptors. As a most well-known thermoresponsive polymer, poly(N-isopropylacrylamide) (PIPAAm), exhibits a lower critical solution temperature (LCST) of approximately 32°C in water.³ Below the LCST, PIPAAm molecule hydrates and has an extended-coil conformation. After increasing temperature above the LCST, the polymer chains collapse and exhibit a globular conformation.⁴ The reversible coil-globule transition of PIPAAm chain can be used for regulating the affinity interactions between bioactive ligands and their cell membrane receptors. Covalently immobilized bioactive ligands such as Arg-Gly-Asp-Ser (RGDS) polypeptide^5,6 and insulin⁷ onto the carboxyl side chains of grafted poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] on tissue culture polystyrene (TCPS) enhance cell adhesion and allow the cells to grow at 37°C. By lowering temperature to 20°C, the confluent cultured cells detach themselves as a contiguous cell sheet from the surface due to the reduction of ligand-receptor interaction by extended PIPAAm chains. Temperature-dependent affinity regulation between other proteins and ligands using PIPAAm chain is also achieved. Yoshizako et al. have investigated the possibility of affinity regulation by both PIPAAm and spacer length for forced-releasing effects.⁸ In case of Cibacron Blue ligand immobilized with shorter spacer-length than the mean size of the extended PIPAAm, captured albumin could be released when temperature is lowered below LCST. Hoffman et al. have immobilized PIPAAm molecule in the vicinity of genetically engineered binding sites of biotin in streptavidin and demonstrated that the binding ability of biotin decreases with increasing temperature higher than LCST.^9,10 They have suggested that the possible mechanism should be due to the blocking of biotin binding site by collapsing PIPAAm molecule.

In contrast to the above-mentioned one-on-one affinity binding between ligand and receptor, the multivalent affinity coordination of specific protein can be also seen in proteoglycan for regulating cell functions. For example, on the cell surfaces, a heparin molecule interacts with various heparin-binding proteins such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), fibronectin, and antithrombin III (AT-III) through multivalent binding. In biology, the interaction between heparin and heparin-binding protein plays important roles in regulating the stabilities and activities of the proteins.¹¹ Therefore, the development of a new material for switching the interaction, and the stabilities and activities of the proteins with heparin by external stimulus such as temperature would promise to provide universal materials for regulating cellular adhesion, proliferation, migration, differentiation, and signal transduction. However, the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins has never been reported.

An accompanying article from Okano and coworkers presented in Biomaterials¹² provides a new concept of functional thermoresponsive cell culture surface for both enhancing a cell proliferation and maintaining cell detachment capabilities by regulating multivalent affinity binding with temperature changes. They have designed heparin-functionalized thermoresponsive surface for multivalent affinity binding with basic fibroblast growth factor (bFGF). Heparin-functionalized thermoresponsive surface can bound active bFGF through surface-immobilized heparin at 37°C, because heparin stabilizes and activates bFGF.¹¹ In addition, confluently cultured cells on heparin-functionalized thermoresponsive surface detach themselves as a cell sheet by lowering temperature. The driving-force of cell detachment is considered to be due to the conformational change of a heparin molecule, which has multiply bound bFGFs, by swelling grafted P(IPAAm-co-CIPAAm) chains. Therefore, the heparin-functionalization of thermoresponsive surface can regulate both the binding and the activation of bFGF by temperature.

In general, protein-immobilization is known to lead to reducing their bioactivities, and it is difficult to preserve a protein in native state and their orientation. By contrast, the immobilization of bFGF through affinity binding with heparin onto surface maintains their activities, and NIH/3T3 cells adhere and grow well accompanied by increasing the amount of affinity-bound bFGF on bFGF-bound heparin-P(IPAAm-co-CIPAAm) grafted surfaces. The internalization of bFGFs within the cells is also inhibited by interaction with surface-immobilized heparin, indicating that the stable binding of bFGFs on heparinized surfaces inhibits the downregulation of FGF receptors and continues to signal the receptors for enhancing cell growth for long-term. Furthermore, the activity of cell growth on bFGF/heparin-P(IPAAm-co-CIPAAm) grafted surface is higher than those of soluble and physisorbed bFGF. The surface of bFGF-bound heparin-P(IPAAm-co-CIPAAm) grafted surfaces can retain 2- to 3-fold of the number cells than the other surfaces.

Interestingly, bFGF on heparin-functionalized thermoresponsive surfaces is released with detaching cell-sheet by lowering temperature to 20°C. Fluorescent microscopy reveals that fibronectin and bFGF are localized on detaching cell sheet, whereas no fibronectin and bFGF are observed on heparin-functionalized thermoresponsive surface where the cell sheet has been removed. Covalent immobilization of growth factors onto cell culture surfaces also gives the long-term activation of intracellular signal transductions.¹³ However, these surfaces consistently stimulate cultured cells without switching the signaling of cell growth. By contrast, heparin-functionalized thermoresponsive surface is a newly designed cell culture system allowing the dynamic switching of cell growth and cell detachment to be regulated by temperature changes.

The use of heparin-functionalized thermoresponsive cell culture surface is useful for clinical application. Heparin-immobilized thermoresponsive cell culture surface can be prepared in large scale by using electron beam (EB)-induced polymerization and covalent grafting, and be stored under dry condition at room temperature, while protein-immobilized surface should be stored under aqueous condition in a refrigerator. In addition, the affinity-binding of bFGF on the surface is quite simple, because bFGFs are only added on heparin-P(IPAAm-co-CIPAAm) grafted surfaces.

Furthermore, heparin-functionalized thermoresponsive surface provides versatile cell culture system, because the surfaces will be able to immobilize other heparin-binding proteins such as VEGF, AT-III, hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species. Therefore, a cultivation technology using heparin-functionalized thermoresponsive cell culture surface has also a potential to achieve the formation of cell sheets using weakly adhering cells such as primary cells onto culture surfaces and to harvest differentiated cells from stem cells with only lowering temperature, which leads to the preservation of receptors on the harvested cellular membrane.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.