Development and characterization of proteinaceous multiphoton-fabricated microstructures with photo-thermal functionality

Abstract

We exploit two-photon laser writing to fabricate 3D biocompatible proteinaceous microstructures (∼1 to 50 𝜇m in lateral size) with tunable elasticity and photo-thermal activity in the near-infrared. Structure printing relies on the photo-crosslinking of the protein bovine serum albumin (BSA, 50 mg/mL) initiated by the Rose Bengal dye (2 mM concentration), whereas photo-thermal functionality is achieved by the dispersion of non-spherically symmetric metallic nanoparticles into the ink.\nAiming at a subsequent application of the fabricated microstructures as platforms for cell growth and stimulation, we carry out a thorough characterization of their mechanical and photo-thermal properties. Preliminary data obtained by AFM indentation have quantified the structures Young modulus in the broad 100-1000 kPa range depending on the BSA concentration. Stiffness is further characterized here by subjecting the fabricated microstructures to steady flow in a microfluidic device, and by quantifying their real-time bending by a conventional transmitted light microscope. In parallel, we focus on the optimization of the photo-thermal activity of the structures. Anisotropic gold nanoparticles, dispersed in the ink, get trapped into the structure during photo-crosslinking and lead to localized heat release upon excitation in the near-infrared. The temperature increment is rapidly (∼1 s) reached and maintained under continuous wave laser irradiation at 800 nm; the amplitude of the temperature variation is quantified as a function of the incident laser power by means of infrared thermography and is correlated to both the structure thickness and the nanoparticles concentration. The resulting spatially confined heat loads could be exploited to induce highly localized responses in cells. In this direction, proteinaceous photo-thermal microstructures can be used to physically induce the differentiation of cells (e.g. neurons or fibroblasts) in a spatially controlled manner.