1 vinyl 2 pyrrolidinone

Microrods (100 µm × 15 µm × 15 µm) and microcubes (15 µm × 15 µm × 15 µm) were fabricated from PEG-DMA as previously described using commercially available materials [24 (link)] and re-suspended in complete media for in vitro studies or sterile saline solution for intra-cardiac injections (Figure 1). Briefly, polyethylene glycol dimethacrylate (PEG-DMA) (MN = 750, Sigma Aldrich, St. Louis, MO) was diluted with Calcium and Magnesium-free 1× Phosphate Buffered Saline (PBS). The photo-initiator 2,2-dimethoxy-2-phenylacetophenone (DMPA) (Sigma Aldrich, St. Louis, MO) solubilized at 100 mg/mL in 1-vinyl-2-pyrrolidinone (Sigma Aldrich, St. Louis, MO) was then added to this mixture in equal volume to the PBS added and vortexed thoroughly. The solution was spun to a 15µm thick layer on a piranha-solution-cleaned silicon wafer (Addison Engineering, San Jose, CA) and exposed through a photomask to a 405 nm UV light source using a Karl Suss MJB3 mask aligner (Suss Microtec, Garching, Germany) to crosslink the desired regions in the shape of microrods (100 µm × 15 µm × 15 µm) or microcubes (15 µm × 15 µm × 15 µm). Microstructures were rinsed, scraped from the surface gently using a cell scraper, and sterilized in 70% ethanol. Before use, microstructures were centrifuged to allow aspiration of the ethanol and re-suspension at the desired number-density in saline solution.

1-vinyl-2 pyrrolidinone (NVP), 2,2-dimethoxy-2-phenylacetophenone (DMPA), eosin Y, ethanol, phosphate buffered saline (PBS), and triethanolamine (TEOA) were purchased from Sigma-Aldrich (St. Louis, MO). I-2959 was provided by Ciba Corporation (Newport, DE). Cell proliferation reagent WST-1 and protease and phosphatase inhibitors were purchased from Roche Applied Science (Indianapolis, IN). Phospho-AKT (Ser473) (p-AKT) antibody was purchased from Cell Signaling Technology (Danvers, MA). AKT1 (559028) antibody was purchased from BD Biosciences Pharmingen (Mississauga, ON, Canada). β-actin (ACTBD11B7) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit antibody conjugated to horseradish peroxidase and goat anti-mouse antibody conjugated to horseradish peroxidase were purchased from Bio-Rad (Hercules, CA).

PEGDA of molecular weights 1000, 4000, 8000, and 10 000 Da were purchased from Monomer-Polymer and Dajac Labs (an MPD Chemicals Company). MAETAC, 2,2-dimethoxy-2-phenylacetophenone, and 1-vinyl-2-pyrrolidinone were purchased from Sigma Aldrich. The photo-initiator solution of 300 mg/mL of 2,2-dimethoxy-2-phenylacetophenone in 1-vinyl-2-pyrrolidinone was prepared using a vortex mixer.
The hydrogels were formed by dissolving the PEGDA into deionized (DI) water using a vortex mixer. The MAETAC was then added and vortexed until sufficiently mixed. Various formulations were created to obtain a hydrogel with the best blend of actuation and biocompatibility. The formulations for each of the samples are listed in Table 1. The photo-initiator solution was added just prior to applying ultraviolet (UV) radiation at a concentration of 100 μL of photo-initiator solution per 1 mL of hydrogel solution. The hydrogel solution was then injected into a rectangular glass mold and cross-linked with UV radiation at a wavelength of 365 nm and intensity of 10 W for 2 minutes. The hydrogels were then placed in phosphate-buffered solution (PBS) overnight to achieve equilibrium swelling. For actuation testing, samples were cut into 20 × 5-mm shapes before actuation.