A Modular Approach to Sensitized Two‐Photon Patterning of Photodegradable Hydrogels

Abstract Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo‐induced degradation of such hydrogel systems is commonly based on the integration of photolabile o‐nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one‐ and two‐photon absorption. Herein we describe a cytocompatible click‐based hydrogel containing o‐nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone‐based small molecule as photosensitizer the efficiency of the two‐photon degradation process can be improved significantly. Biocompatibility of both the improved two‐photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.

3 functionalized with thiol groups using 1 H-NMR analysis (D2O) by comparing the integrals of the -CH2CH2SH side chain methylene peaks at 2.58 and 2.73 ppm with the N-acetyl moiety of HA at 1.97 ppm ( Figure S1b).

S1.3 Gel formation of PEG-HA-SH hydrogel via Michael thiol-ene reaction
HA-SH was dissolved in Dulbecco's modified Eagle's medium (DMEM) or Endothelial cell Growth Medium (EGM-2, Lonza) and neutralized with aqueous NaOH (0.1 M) to form an 18 mg ml -1 solution. PEG-(oNB-A)2 was dissolved in PBS giving a solution of 150 mg ml -1 . The solutions were combined with 1:1 stoichiometry of functional groups at 6.7 wt% total macromer concentration and used immediately. Hydrogels based on HA-SH with different DS were prepared analog but resulting in a different total macromer concentration (HA-SH DS = 40%: 5.5 wt%).

S1.5 Photo-rheology measurement of photodegradable hydrogels
The time required for network formation was estimated by measuring the shear storage modulus G' using a MCR-302 WESP rheometer with a P-PTD 200/GL peltier glass plate and a PP25 measuring system (Anton-Paar). Hydrogel samples were prepared as described in the above sections. For control experiments involving P2CK the linker PEG-(oNB-A)2 was dissolved in an adequate amount of P2CK solution (1.0 mM) and PBS so that the resulting hydrogel formulation contained 0.1 mM P2CK. Immediately after mixing of the components 55 µl of the solution were placed at the center of the glass plate. The measurements were conducted at 20 °C using a plate-plate measuring system (Ø = 25 mm, gap size = 50 µm). The formulations were sheered with a strain of 1% and a frequency of 1 Hz. Gelation was assumed to be complete when G' reached a plateau. Photo-degradation of hydrogel samples was induced by UV-irradiation projected via a waveguide (35 min, 320-500 nm, ~20 mW cm -2 at the measuring platform) from the underside of the glass plate using an OmniCure® S2000 Spot UV Light Curing System (Excelitas Technologies). [5] Alternatively, an OmniCure® LX400 LED UV Spot Curing System with a 460 nm LED head was used (Excelitas Technologies) with a specific power of ~17 mW cm -2 at the measuring platform.

S1.6 Preparation of P2CK solutions
Solutions of P2CK at concentrations ranging from 1.00 to 0.01 mM were freshly prepared before each experiment by dilution of a stock solution in PBS (10 mM) with either adequate cell culture medium or PBS.

S1.7 Two-photon micropatterning of channels in photocleavable hydrogels
Aliquots (40 µl) of the mixed PEG-HA-SH hydrogel precursor solutions were drop casted onto methacrylized glass-bottom µ-dishes (35 mm, Ibidi GmbH, Germany) and allowed to gel at room temperature for 90 min in accordance to the gelation duration observed by rheological measurements. The solidified hydrogel droplets were immersed in either DMEM or solutions of P2CK and swollen for ~5 h at room temperature before one half of each sample was cut away using a scalpel to generate a sharp edge. Aliquots (27 µl) of the mixed 4armPEG-SH hydrogel precursor solutions were formed in cylindrical silicon molds (d = 6 mm, h = 0.7 mm) between glass-bottom µ-dishes and hydrophobized cover glass. After 40 min the molds were carefully removed and the 4armPEG-SH hydrogels were swollen in PBS overnight before they were treated with P2CK as described above.
Microfabrication was performed by means of two-photon degradation. Details of the experimental setup have been reported previously. [6] Briefly, the setup is based on a femtosecond laser (MaiTai DeepSee, Spectra Physics) operating at 800 nm, with a pulse length of 70 fs after the objective (C-Achroplan 32x/0.85 W, ZEISS). Parallel channels with rectangular cross sections (l = 300 µm, A = 20 µm x 50 µm) were fabricated starting from the edge into the bulk of the hydrogel at a height of ~60 µm (~200 µm in case of 4armPEG-SH hydrogels) above the glass plate with varied mean laser power per channel (10-100 mW, 10 mW steps) and a constant scanning speed of 200 mm/s (hatch distance: 0.1 µm, z-layer distance: 0.5 µm). The samples were washed with PBS twice and then soaked in a solution of FITC-dextran (1 mg ml -1 , Mw ~2,000 kDa, TdB Consultancy AB, Sweden) in PBS at room temperature overnight. Channels were visualized by laser scanning microscopy (LSM 700, ZEISS).

S1.8 Atomic force microscopy (AFM) indentation testing of two-photon micropatterned cuboids in PEG-HA-SH hydrogel
A droplet (10 µL) of PEG-HA-SH hydrogel was prepared in a cylindrical mold (d = 6 mm, h = 0.2 mm) on methacrylized glass bottom µ-dishes. To ensure a smooth surface no lid was used for molding. After 90 min the mold was removed and the hydrogel sample was swollen in PBS for 2.5 h. Cuboids (A = 100 x 100 µm 2 ) with an approximate depth of 100-150 µm were two-photon micropatterned from the upper surface into the hydrogel starting above the surface.
Subsequently, PBS was exchanged for a 0.1 mM solution of P2CK in PBS and the sample was maintained at room temperature for 1 h. Again, cuboids were micropatterned into the hydrogel at the same parameters as before. Thereafter, the hydrogel was swollen in PBS overnight. A NanoWizard® ULTRA SpeedA AFM system (JPK Instruments AG, Germany) equipped with an inverted optical microscope (Axio Observer.D1, ZEISS) was used for AFM experiments. The micromechanical assessment was performed via AFM cantilever-based microindentation experiments using a silicon nitride rectangular cantilever (0.0196 N m -1 measured spring constant; MSNL, Bruker) equipped with a colloidal probe of 4.75 μm in radius.
The thermal noise method [7] was used to calibrate the cantilever spring constant. The deflection sensitivity was obtained by performing 16 force measurements on the glass surface next to the sample. [8] After calibration, the cantilever was fabricated with the colloidal probe [9] and its diameter was measured as previously described. [8] In total, 50-60 force curves were recorded at 1 nN maximum load and 2 μm s -1 of z-displacement speed per irradiated section. The Oliver-Pharr method was used to analyze the force curves and estimate the indentation modulus, as previously described, in a custom MATLAB script (v R2015b, MathWorks, USA). [8] For the analysis, the Poisson's ratio of the hydrogel was taken as 0.5. The relative height measurement results are presented as mean ± standard deviation. The indentation modulus results are presented as mean ± standard error of the mean.
Cells were maintained in an incubator (5% CO2, 37 °C) and the medium was exchanged every second day. Cell spheroids were formed in 2% agarose molds (MicroTissues® 3D Petri Dish®,

S1.10 Evaluation of P2CK cytocompatibility
The cytocompatibility of two-photon sensitizer P2CK was evaluated using PrestoBlue® cell viability reagent (Thermo Fisher Scientific). Two 96-well plates were seeded with 5,000 cells per well and maintained in the incubator overnight to allow cells to attach. Next day, cells were

S1.13 Evaluation of optical parameters
To fully describe an experiment with multiphoton activation, parameters such as laser power after the objective, numerical aperture of the objective, wavelength, scanning speed, hatch and z-layer spacing need to be stated. Unfortunately, in many publications not all of these parameters are presented, which makes it very difficult to compare experiments. Therefore, we would like to introduce this parameter set as a guideline for future publications.
The peak intensity IPeak is an important factor, because it allows the direct comparison of two different multiphoton polymerization systems. As an example, objectives with different numerical aperture NA perform the same, if the peak intensity is the same, and the average power has been adjusted correspondingly. The peak intensity is obtained by dividing peak power PPeak by the beam area and multiplied with the correction factor of 2 for a Gaussian beam          The cell viability of ASCs exposed to P2CK for 3 h and 24 h was examined by PrestoBlue® metabolic activity assay. Data are shown as mean + standard deviation (n = 8 replicates per group) and were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.   The cell viability of MG-63s exposed to P2CK for 3 h and 24 h was examined by PrestoBlue® metabolic activity assay. Data are shown as mean + standard deviation (n = 8 replicates per group) and were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.