Submicron Multiphoton Free-Form Fabrication of Proteins and Polymers:  Studies of Reaction Efficiencies and Applications in Sustained Release

Abstract

Nonlinear multiphoton photo-cross-linking and photopolymerization of proteins and polymers in solution have been used to direct the three-dimensional assembly of micron scale objects. Two aspects of fabricated proteinatious matrixes are examined in this paper: the efficiency of protein photopolymerization and the application of fabricated matrixes as sustained release devices. The efficiency of photoactivated cross-linking of the proteins bovine serum albumin and fibrinogen, using rose bengal, have been determined and found to vary with photosensitizer concentration. This concentration dependence suggests that the mechanism for protein cross-linking is a direct hydrogen transfer between an amino acid residue of the protein and the dye molecule itself. A comparison of the surface structure of single and multiple protein oligomers is undertaken and shown to vary significantly depending on fabrication materials. Alkaline phosphatase bioactivity, upon entrapment in a protein structure, is maintained. The properties of fabricated protein matrixes as sustained release devices is also examined. The rates of diffusion of fluorescently labeled dextrans (10 and 40 kDa) from an optically fabricated BSA matrix vary with molecular weight and are linear with cross-link density. The half-life of release of 10 kDa dextran-TMR from a BSA micron scale structure is less than or equal to 6 min while 40 kDa dextran-TMR halflife of release is 25 min. Finally, rhodamine 610, a typical drug size molecule, was entrapped in an acrylamide structure, and its release is found to be diffusion-limited with half-lives of 10-31 min, depending on cross-link density.

Multiphoton fabrication of chemically responsive protein hydrogels for microactuation

“…We and others have shown that direct-write protein matrices can retain ligand-binding and catalytic functionality similar to that of their component proteins (23)(24)(25)(26)(27), implying that native electrostatic and hydrophobic interactions governing protein conformation remain significantly intact within microstructures. However, at pH extremes, unfavorable electrostatic interactions can disrupt the native state and result in protein unfolding (31,32).…”

“…High concentrations of protein in solution facilitate fabrication of robust microstructures. However, fabrication can be accomplished using protein solutions at concentrations as low as Ϸ1-10 ng/ml (27). Although some proteins undergo multiphoton crosslinking more efficiently than others, a broad range of proteins can be used (e.g.…”

“…In addition to having the potential to undergo shape and hydration changes in response to chemical triggers, proteins contain a large number of weak acids and bases, similar to polymers used in pH-responsive hydrogels (5). Importantly, matrices composed of functional photocrosslinked proteins can be localized in 3D microenvironments by using multiphoton fabrication (23)(24)(25)(26)(27). Thus, multiphoton-fabricated protein structures could provide high-resolution 3D control over the topography of a stimuliresponsive material-facilitating greater mechanical functionality and incorporation of responsive hydrogels into more complex 3D devices-while potentially providing specificity (e.g., through ligand binding) over volumetric responses.…”

Introducing Defects in 3D Photonic Crystals: State of the Art

“…MPP was first developed by Strickler and Webb, [62] and has now been used to create a range of high-resolution 3D free-form structures, including microchannels, [63][64][65] cantilevers, [66][67][68] microgears, [69,70] sub-micrometer oscillators, [71] hydrophobic atomic force microscopy tips, [72] and PhCs. [59,67,[73][74][75][76][77][78][79][80][81] Briefly, MPP utilizes the nonlinear nature of the multiphoton excitation process to only excite dye molecules in a very small volume around the focal point, with dimensions on the order of the resolution limit.…”

Three‐Dimensional Bioactive and Biodegradable Scaffolds Fabricated by Surface‐Selective Laser Sintering

“…Hence, the starting materials limit the range of biological responses that can be obtained in the final product. Nonlinear multiphoton crosslinking and polymerization of proteins and monomers (e.g., alkaline phosphatase and acrylamide) can be achieved using femtosecond laser systems and such [6] processes have been successfully exploited for fabrication of 3D sustained-release devices on the subcellular biological scale by microstereolithography. [7] However, the small size (ca.…”