Photo-click living strategy for controlled, reversible exchange of biochemical ligands

Abstract

A novel addition-fragmentation-chain transfer capable allyl sulfide functionalized PEG hydrogel is reported to allow controlled, reversible exchange of biochemical ligands. The exchange of biochemical ligands is achieved without permanent consumption of reactive functionalities. Demonstrated is the ability to exchange biochemical ligands multiple times using cytocompatible 720 nm two-photon light.

Keywords: addition fragmentation chain transfer; hydrogels; photo-patterning; thiol-ene reaction.

Figure 1

A) Structures of monomers used to form the hydrogel networks. 1: 4-arm-poly(ethylene glycol)-tetra azide. 2: 2-methylene-propane-1,3-bis(thioethyl 4-pentynoate). 3: Benzoic acid-3,5-bis(4-pentynoate). B) Mechanism of patterning fluorescently tagged CRGDS to the allyl sulphide functionalized hydrogel. C) Hollow cage pattern formed by exposing specific regions in –x and –y directions to 720 nm 2-photon light and rastering the focal point through z-direction. Thiol-ene reaction is initiated by I2959 initiator resulting in tethering of AF555-CRGDS only in the exposed regions. D) Amount of the peptide tethered to the network can be regulated by controlling the laser power and laser scan speed. E) Corresponding fluorescent patterns formed by controlling the laser scan speed and laser power.

Figure 2. Photoclick living strategy allows reversible exchange of biochemical ligands

A) Mechanism of replacement of a thiol-containing compound on allyl sulphide functional group. B) Schematic of replacement of biochemical ligand. C–K) Demonstration of reversible exchange of thiol containing peptides. (C–E) 250µm square pattern of AF₅₅₅AhxRGDSC. (F–H) Buffalo logo was formed by replacing AF₅₅₅AhxRGDSC with AF₄₈₈AhxRGDSC resulting in appearance of green fluorescence and disappearance of red fluorescence only in the exposed regions. (I–K) Demonstration of further replacement on living allyl sulfide: Letters ‘CU’ inside the buffalo logo were exposed to 720 nm light to photo-exchange with non-fluorescent AhxRGDSC peptide. Photo-exchange of peptides was confirmed by removal of green fluorescent (J) only at the exposed regions. Scale bar = 100 µm.

Figure 3. Kinetics of exchange of biochemical ligands in AFCT capable hydrogels

A) Amount of AF₅₅₅AhxRGDSC remained attached to the network after the photo-exchange reaction as a function of dosage at different laser scanning speeds. B) Amount of AF₄₈₈AhxRGDSC photo-coupled to the network due to the exchange as a function of dosage at different pixel dwel times. C–E) Simultaneous generation of opposing gradient patterns of two different biomacromolecules. (C) AF₅₅₅, (D) AF₄₈₈RGDS and (E) combined. Gradient pattern is formed by exposing a uniform 250 µm square pattern to radially increasing laser power at pixel dwell time 36.67 µ/sec²

Figure 4. ‘Living’ strategy allows cytocompatible manipulation of biochemical environment of the gel

A) 0.5 mM of rectangular RGD pattern was formed and seeded with hMSCs. B) hMSC stained with cell tracker red attached to the rectangular pattern. C) A smaller region was exposed to 720 nm light to allow for exchange of fluorescent-RGD with non-fluorescent RDG peptide. The photopatterning did not affect the cellular staining. Scale bar = 25 µm.

Scheme 1

Mechanism of addition fragmentation chain transfer of an allyl sulfide functional group upon attack by a thiyl radical.