Wavelength-controlled photocleavage for the orthogonal and sequential release of multiple proteins

Abstract

On the right wavelength: Photolabile molecular units that undergo photocleavage under light of different wavelengths can be used for the independent release of different dyes/proteins from a single, preloaded storage hydrogel. The controlled release of each protein allowed them to be delivered sequentially and at experimenter-determined times.

Keywords: dyes/pigments; hydrogels; photocontrolled release; protein delivery; proteins.

Figure 1

Wavelength dependent orthogonal photodegradation: a) Photocleavage of nitrobenzyl (NB) and coumarin methylester (CM) molecular systems: 1 and 2 and their corresponding photocleaved products 3 and 4; Comparative photodegradation of 1 and 2 at (b) 365 nm and (c) 405 nm for different times of light exposure. d) Absorbance spectra of 1 (0.4 mM) and 2 (0.4 mM) dissolved in acetonitrile/PBS mixture (8:2).

Figure 2

(a) Chemical structures of rhodamine B tethered nitrobenzyl azide 5 and fluorescein tethered coumarin azide 6; (b) PEG polymeric precursors that produce SPACC hydrogel networks: 4-arm PEG-tetra DBCO 7 and 4-arm PEG-tetraazide 8; (c) Schematic representation of light-wavelength regulated selective release of dye molecules from hydrogel networks; Dye release studies upon exposure to light sources of (d) 365 nm (10 mW/cm2) for 5 mins and (e) 405 nm (10 mW/cm2) for 20 mins. Gray bars indicate the light exposure.

Figure 3

Daily release of BMP-2 and BMP-7 upon exposure to light of (a) 365 nm (5 mW/cm²) and (b) 405 nm (5 mW/cm²) for varied times; (c) Sequential release of BMP-2 and BMP-7 upon exposing to both 405 and 365 nm light but at different time points; d) Relative ALP activity for hMSCs seeded on hydrogels that covalently contained both BMP-2 and BMP-7 in equal amounts (10ng/ml) and exposed to 365 nm (for 6 mins) and 405 nm (for 12 mins) separately and also to both but sequentially, i.e., 405 first for 12 mins and then 365 for 6 mins (405 to 365 nm, 5mW/cm²); native BMPS: cells seeded on PEG hydrogels and exposed to BMP-2 and BMP-7 (10ng/ml) but at day 1 and day 4 respectively; no light: not exposed to any light but gel covalently contained both BMPs; no BMPs: cells seeded on hydrogel that did not contain any proteins.

Scheme 1

Incorporation of photocleavable azides onto BMP-2 and BMP-7 proteins respectively: (i) Traut's reagent, pH: 7.0, Phosphate buffer (PBS), rt; (ii) 7 (for BMP-2) or 8 (for BMP-7), PBS, pH: 7.0, rt.