The extracellular matrix (ECM) plays a role in influencing cell behavior. In recent years it has become evident that cell biological functions are also closely linked to the three-dimensional (3D) spatial distribution of ECM protein [1,2], and the geometric and mechanical constraints of their microenvironment. Two-photon polymerization (2PP) prototyping technique enables the microfabrication of static and dynamic 3D structures, which can be selectively functionalized with bioactive molecules (e.g., ECM proteins, oligonucleotides, etc.) [2,3]. In turn, these enable cells anchorage [4], thus mimicking the physiological extracellular microenvironment [5]. The patterning of 3D structures for protein immobilization has already been the subject of various complex approaches, which usually include chemical modification of the resist using photo-reactive molecules, such as photoenols [2,4].
The presented procedure demonstrates the possibility of biofunctionalizing 2PP-printed microstructures realized using chemically unmodified commercial photoresists, via light-activated click reactions. Specifically, two different functionalizable resists based on trimethylolpropane ethoxylate triacrylate (TPETA) and pentaerythritol triacrylate (PETA) were used for the fabrication of (sub)micrometric structures using 2PP. The structures were functionalized with N-biotinoyl-N'(6maleimidohexanoyl) hydrazide (Maleimide-Biotin, Mal-Bt) through UV laser irradiation (405 nm) and subsequently incubated with streptavidin-TRITC (Figure 1A). The red fluorescent signal (Figure 1B) is only visible in selected areas within the structures, indicating that only those areas exposed to light have been successfully functionalized. Figure 1C-D shows the relationship between the relative fluorescence intensity obtained for different exposure conditions and the relative dose received by the different samples.
Results suggest that the described procedure can be used for the realization of free-shape responsive 3D microscaffolds capable of selectively anchoring cells, to stimulate them in a physiological-like microenvironment.

References

[1] M. J. Randall, Front. Bioeng. Biotechn., 2018.

[2] B. Richter, Adv. Mater., 2017.

[3] M. T. Raimondi, J. Appl. Biomat. Funct. Mater., 2012.

[4] E. D. Lemma, ACS Appl. Mater. Interfaces, 2023.

[5] E. D. Lemma, Trends Biotechn., 2019.