New Photoactivators for Multiphoton Excited Three-dimensional Submicron Cross-linking of Proteins: Bovine Serum Albumin and Type 1 Collagen¶†

Abstract

We report the synthesis and optical characterization of two new photoactivators and demonstrate their use for multiphoton excited three‐dimensional free‐form fabrication with proteins. These reagents were developed with the goal of cross‐linking Type 1 collagen. This cross‐linking process produces structures on the micron and submicron size scales. A rose bengal diisopropyl amine derivative combines the classic photoactivator and co‐initiator system into one molecule, reducing the reaction kinetics and increasing cross‐linking efficiency. This derivative was successful at producing stable structures from collagen, whereas rose bengal alone was not effective. A benzophenone dimer connected by a flexible diamine tether was also synthesized. This activator has two photochemically reactive groups and is highly efficient in cross‐linking bovine serum albumin and Type 1 collagen to form stable, robust structures. This approach is more flexible in terms of cross‐linking a variety of proteins than by traditional benzophenone photochemistry. The photophysical properties vary greatly from that of benzophenone, with the appearance of a new, lower energy absorption band (λmax∼370 nm in water) and broad, visible emission band (∼500 nm maximum). This absorption band is highly solvatochromic, suggesting it arises, at least in part, from a charge transfer interaction. Collagens are typically difficult to cross‐link photochemically, and the results here suggest that these two new activators will be suitable for cross‐linking other forms of collagen and additional proteins for biomedical applications such as the de novo assembly of biomimetic tissue scaffolds.

Multiphoton excited fabricated nano and micro patterned extracellular matrix proteins direct cellular morphology

“…The primary fibrinogen antibody (American Diagnostica, Greenwich, CT), the fluorescent secondary antibody Alexa fluor 488 F(ab′)2 fragment of goat antimouse IgG (Molecular Probes, Eugene, OR), and the vinculin primary antibody (Sigma) were used as received. The benzophenone dimer (BPD) photoactivator was synthesized as described previously 36…”

“…Fibronectin (1 mg/mL) is crosslinked via 3‐photon excitation of the BPD at 780 nm. This reagent inserts into either carbonhydrogen, or carbon carbon bonds, and becomes part of the crosslinked network 36. This reagent is particularly effective for crosslinking minimally soluble ECM proteins, and was initially developed in our laboratory for crosslinking collagen 36.…”

“…This reagent inserts into either carbonhydrogen, or carbon carbon bonds, and becomes part of the crosslinked network 36. This reagent is particularly effective for crosslinking minimally soluble ECM proteins, and was initially developed in our laboratory for crosslinking collagen 36. Additionally, preliminary data suggested that superior fibroblast adhesion was obtained on fibronectin crosslinked with the BPD rather than RB.…”

Visible-Light-Mediated Modification and Manipulation of Biomacromolecules

“…319,320 Particularly when coupled with the exquisite three-dimensional resolution of laser irradiation in the 2PE regime, protein photocross-linking has also been used to fabricate microstructures on the (sub)micrometer scale from various proteins. 310,315,321,322 2.3.4. Chromophore-Assisted Laser Inactivation.…”

“…Deiters and co-workers subsequently applied 313 to control DNA recombination in zebrafish embryos by generating a caged Cre recombinase enzyme whose activity was restored upon visible-light irradiation (405 nm). 543 Following their report highlighting NDBF as a cysteinecompatible PPG (see Scheme 90), 537 Distefano and coworkers developed the 6-bromo-7-hydroxy-3-methylcoumarin-4-ylmethyl (mBhc) caging group, which was applied to obtain mBhc−Cys (315). Coumarin-based PPGs have several advantages over NDBF as cysteine cages: they are relatively straightforward to synthesize, more water-soluble, and also fluorogenic.…”