Sensitizer-enhanced two-photon patterning of biomolecules in photoinstructive hydrogels

Synthesis of TPA-trisNTA

Fmoc-protected DEAC β-amino acid was synthesized in six steps and characterized by nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HR ESI-MS). TPA-trisNTA (trisNTA-AC-DEAC-GHHHHH-OH) was synthesized through microwave-assisted Fmoc solid-phase methodology using the Liberty Blue microwave peptide synthesizer (CEM, Germany) and starting from a preloaded Fmoc-His(Trt)-Wang (Sigma Aldrich, Germany) resin. All reactions except for coupling of the Fmoc-protected DEAC β-amino acid (single coupling) were done twice with 0.2 M of Fmoc-protected amino acid, 0.5 M N,N’-diisopropylcarbodiimid, 0.5 M 1-hydroxybenzotriazole monohydrate in dimethylformamide (DMF). Fmoc-deprotection was performed with 20% (v/v) piperidine in DMF. O^tBu-protected carboxy-trisNTA was manually coupled on the N-terminal glycine using (1-cyano-2-ethoxy-2-oxoethylidenamino-oxy)dimethylamino-morpholino-carbenium-hexafluoro-phosphat (COMU) and N,N-diisopropylethylamine (DIPEA) in DMF. Peptide cleavage/deprotection was achieved using 95% trifluoroacetic acid (TFA), 2.5% H₂O and 2.5% 1,2-ethanedithiol followed by two precipitation cycles (50 mL diethyl ether, 0 °C). The crude peptide was purified via preparative reverse-phase high-performance liquid chromatography (RP-C₁₈-HPLC) using a 30 min gradient (5–70% of acetonitrile and 0.1% TFA in H₂O) and lyophilized (Supplementary Methods 1.3, Supplementary Fig. 4). TPA-trisNTA purity was confirmed by HR ESI-MS. The complete synthetic details and characterization of TPA-trisNTA and Fmoc-protected DEAC β-amino acid are given in the Supplementary Methods. For NMR and ESI spectra see Supplementary Figs. 22–36.

Photolysis reaction of the DEAC ß-amino acid in solution

50 µL aliquots of a stock solution of Fmoc-protected DEAC β-amino acid in HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl pH 7.2) were illuminated at 405 nm (185 mW/cm², Thorlabs LED system). To study the kinetics of photolysis, samples were taken at different time points (0, 5, 10, 15, 30, 45, 60, 300, and 900 s). Photocleavage of Fmoc-protected DEAC β-amino acid was assessed by analytical RP-C₁₈-HPLC (20 min gradient, 5–100% of acetonitrile and 0.1% TFA in H₂O, Supplementary Methods 1.3, Supplementary Table 2), revealing conversion to uncaged DEAC β-amino acid with a different retention time (Supplementary Fig. 6). Photolysis efficiency was determined by evaluating the integral of the Fmoc-protected DEAC β-amino signal (t_R = 17.0 min) as well as of the uncaged product (t_R = 12.0 min). The photolysis kinetics were fitted with a mono-exponential function to determine the lifetime t_1/2, indicating a fast one-step photoconversion.

Two-photon absorption spectrum of TPA-trisNTA

Two-photon excitation fluorescence (TPEF) measurements were performed using a tunable Ti:Sa laser (Tsunami, Spectra-Physics, USA) with a pulse duration of 150 fs and a 80 MHz repetition rate. The TPEF-signal was coupled into a spectrograph (SpectraPro 300i, Acton Research Corp., USA), equipped with a CCD-camera (EEV 400_1340F, Roper Scientific, USA). The excitation was adjusted to an average energy of 100 mW and the pulses were tightly focused on the sample compartment. The concentrations of the TPA-trisNTA and coumarin 307 were adjusted to 100 µM in a final volume of 300 µL. Coumarin 307 was used as reference, and values for calculation were taken from Xu et al.⁵⁴. To obtain the two-photon absorption spectrum, the two-photon absorption action cross sections in the range of 770–870 nm were determined. After baseline and detector correction of the fluorescence spectra, the integrals were computed to obtain I_F(X) and I_F(R). For calculations the following equation was used:

with σ₂ = two-photon absorption cross section, ϕ_F = fluorescence quantum yield, X = TPA-trisNTA, R = coumarin 307, I_F = fluorescence intensity, c = concentration, η = refractive index of the solvent. The one-photon fluorescence quantum yield was assumed to equal the two-photon fluorescence quantum yield. The refractive indices of the samples (at 20 °C, Vis-NIR range) were determined to be η(R) = 1.33 (in MeOH) and η(X) = 1.33 (in H₂O).

Two-photon power dependency measurement of TPA-trisNTA

To validate the two-photon absorption of TPA-trisNTA, a scan of the fluorescence intensity over a series of excitation energies (20–100 mW) was performed at a specific wavelength (800 nm). The count rate of the fluorescence response was logarithmically plotted against the logarithm of the excitation energy. A slope of 1.93 ± 0.02 was obtained, implicating an almost quadratic power dependency of the two-photon response. According to Bradley et al. deviations from a strict quadratic power dependency can occur due to heating effects or competing non-linear processes⁵⁸.

Confocal imaging

Imaging was performed using a confocal laser-scanning microscope (LSM 880 AxioObserver, Carl Zeiss Microscopy, Germany), and images were taken with the Plan-Apochromat 20x (NA 0.8) and/or the Plan-Apochromat 63x/Oil (NA 1.4) objective. The following laser lines were used for excitation: 405 nm (diode laser) for Hoechst33342; 488 nm (argon laser) for green fluorescent protein (GFP-His₆); and 633 nm (helium-neon laser) for His₆-AF647 and Annexing V-AF647. All probes were imaged in HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C) or in case of live cells incorporated in the gel, the hydrogel was covered with Live Cell Imaging Solution (Invitrogen). The imaging process was controlled with the Zeiss Zen Black software. Image processing and evaluation was done with Fiji⁵⁹.

Formation of TPA-trisNTA functionalized hydrogels

Gridded ibidi imaging dishes (µ-Dish 35 mm, high Grid-500, ibidi, Germany) were used for hydrogel preparation. For protein photopatterning, hydrogels were prepared from a commercial slow gelling 3-D Life PVA-PEG Hydrogel Kit (Cellendes, Germany). Gel formation was performed by mixing 0.6 µL HBS buffer pH 7.2 with 0.98 µL H₂O and 1.20 µL maleimide-PVA (Supplementary Fig. 8). Then, 1.52 µL of a 790 µM TPA-trisNTA solution (aq.) was added and immediately mixed via pipetting and incubated for 5 min at RT in the dark. After a quick spin down, the reaction mixture was added to 1.74 µL dithiol-linker placed in the middle of an ibidi imaging dish and quickly mixed by pipetting. The network formation was allowed to proceed for 10 min in the dark. The solidified hydrogel was equilibrated with 1 mL HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C).

Laser-assisted photopatterning in hydrogels

For photostructuring by one- or two-photon excitation, a confocal laser-scanning microscope (LSM 880 AxioObserver, Carl Zeiss Microscopy, Germany) equipped with a Plan-Apochromat 63x/Oil (NA 1.4) objective, a 405 nm LED diode, and an ultrafast Ti:Sa Chameleon laser (Coherent Inc., Santa Clara, USA) with a pulse duration of 140 fs, a 80 MHz repetition rate and maximal output of 2.5 W tuned to 800 nm was employed for localized illumination. The user-defined ROI scanning mode and bleaching option were used to precisely control arbitrary patterns in 3D. The laser output power of the Ti:Sa Chameleon laser was precisely adjusted before each experiment to match 0.5, 1.5, 3.1 and 5.5 mW (corresponding to 0.3–0.7–1.2–2.2% of maximal laser power). The illumination time was 6, 12, 18, 24, or 30 s, equaling the sum of 500–2500 iterations. The scan speed was fixed at 1.54 μs/pixel. Photoactivation of hydrogels was performed in HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C). The illumination process was controlled with the Zeiss Zen Black software.

TPA-trisNTA mediated POI immobilization to hydrogels in 3D

Laser-treated hydrogels were washed five times with 2 mL HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C) prior to His-tagged POI tethering. All steps were performed at room temperature and protected from light. For POI patterning, the gel was covered with 1 mL of 10 mM NiCl₂ in MQ water and incubated for 30 min followed by five times washing with HBS buffer (2 mL). Visualization of written structures was realized by incubation with 1 mL of 300 nM His₆-GFP in HBS buffer or 300 nM His₆-AF647 (HHHHHHSGGGSGGG-C^AF647-A-NH₂) in HBS buffer. After 30 min, the hydrogel was washed with HBS buffer (50 mL) by gently shaking for 30 min. Finally, the hydrogel was covered with 1 mL HBS buffer and subsequently imaged via CLSM (LSM 880 AxioObserver, Carl Zeiss).

Global photolithography of hydrogels

For photopatterning of large areas, hydrogels were exposed to UV light (λ = 405 nm LED lamp, 185 mW/cm², 1 min) through a quartz mask with various sized chrome patterns on top of the gel. After illumination, the hydrogels were washed five times with HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C). POI binding and patterning was performed with His₆-GFP after incubation with 10 mM NiCl₂ in MQ. POI assembly was visualized by CLSM (LSM 880 AxioObserver, Carl Zeiss).

Cell viability assessed in cell-laden hydrogels

To analyze cell viability throughout encapsulation in hydrogels, a live-cell annexin V staining was performed to report on apoptotic cells. HeLa Kyoto cells (1 × 10⁶ cells/mL) were washed and suspended in 100 µL annexin V binding buffer (Invitrogen). After adding 25 µL of annexin V-AF647 conjugate (Invitrogen) and one drop of NucBlue Live Ready Probes (ThermoFisher), cells were incubated for 15 min at room temperature and then washed once again with annexin V binding buffer. For hydrogel encapsulation, 10 µL of the HeLa cell suspension were mixed with 3.0 µL HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C), 11.3 µL H₂O, 2.5 µL maleimide-PVA, 0.8 µL of a 20 mM TPA-trisNTA solution (aq.) and 3.0 µL dithiol-linker (Supplementary Table 4). After mixing via pipetting, hydrogel network formation was allowed to proceed for 10 min, followed by covering with 1 mL Live Cell Imaging Solution (Invitrogen). Cell viability analysis was performed by CLSM (Supplementary Fig. 16). Hoechst33342 stained nuclei were excited with 405 nm diode laser and HeLa cells in an early apoptosis state were displayed via the AF647 signal of the annexin V-AF647 conjugate and excited with the 633 nm laser. 708.5 µm × 708.5 µm images were taken. The total amount of cells indicated by the Hoechst33342 signal and the number of AF647-stained cells were counted with Fiji multipoint tool⁵⁹. Determining the ratio between Hoechst33342- and AF647-stained cells revealed that 91 ± 1% of the encapsulated cells were vital. All experiments were performed in triplicates and error bars indicate the s.d. For general cell culture maintenance, see Supplementary Methods 1.10.

Preparation of cell-laden hydrogels for photopatterning of proteins

Y2R expressing HeLa cells were detached with 0.05% trypsin/0.02% EDTA/PBS (GE Healthcare) 16 h post receptor induction, centrifuged (300 × g for 3 min) and solubilized in 200 µL Life Cell Imaging Solution (Invitrogen). For hydrogel formation, 10 µL of the HeLa cell suspension were mixed with 3.0 µL HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C), 11.3 µL H₂O, 2.5 µL maleimide-PVA, 0.8 µL of a 20 mM TPA-trisNTA solution (aq.) and 3.0 µL dithiol-linker. After mixing via pipetting, hydrogel network formation was allowed to proceed for 10 min. Subsequently, the hydrogel was covered with 1 mL Life Cell Imaging Solution. For mask lithography, hydrogels were exposed to UV light (λ = 405 nm LED lamp, 185 mW/cm², 1 min) through a quartz mask with lattice designed chrome patterns on top of the gel (Supplementary Fig. 18). Laser lithography by one- or two-photon excitation was conducted on a confocal laser-scanning microscope utilizing the 405 nm LED diode (maximum output of 4.5 mW) or the ultrafast Ti:Sa Chameleon laser (Coherent Inc., Santa Clara, USA) tuned to 800 nm and a Plan-Apochromat 63x/Oil (NA 1.4) objective in Life Cell Imaging Solution at 37 °C. The written ROIs were visualized by binding of His₆-mCherry (300 nM) or via following His₆-Y₂R^mEGFP assembly at the activated cell hemisphere (Supplementary Fig. 19, Supplementary Fig. 20).

Photoenhancement effect of two-photon sensitizers in hydrogels

Photopatterning by two-photon excitation was conducted on a confocal laser-scanning microscope utilizing the ultrafast Ti:Sa Chameleon laser (Coherent Inc., Santa Clara, USA) tuned to 800 nm and a Plan-Apochromat 63x/Oil (NA 1.4) objective. Before each experiment, the laser intensity of the Ti:Sa Chameleon laser was precisely adjusted to match the given laser powers. 20 rectangular ROIs (15 µm width) were written into the gel with stepwise increased laser power (0.5, 1.5, 3.1, and 5.5 mW) or illumination time (6, 12, 18, 24, 30 s). The scan speed was fixed at 1.54 μs/pixel. Precise control of the laser dosage assigned to each ROI allowed generating photopatterns of different densities. Photoactivation of hydrogels was performed in HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C), in presence of ATTO390 (50, 100, 500 µM in HBS buffer) or of RB (50, 100 µM in HBS buffer). After rinsing with HBS buffer, Ni(II)-loading (10 mM in MQ water) and washing with HBS buffer, His₆-AF647 (300 nM) tethering was performed. Via CLSM, z stacks (35 slices, z step width 1 μm) of the written ROIs were recorded. Imaging conditions (laser power, detector amplification and pinhole) were kept constant and applied to all photopatterned hydrogels. After background subtraction, z slices of each ROI were summed up and quantified by determining the total integrated fluorescence density of each voxel (Supplementary Fig. 11, Supplementary Fig. 12). For comparison, the total integrated fluorescence densities obtained under equal conditions were averaged (Supplementary Fig. 13, Supplementary Fig. 14). Each experiment was performed in triplicate or quadruplicate, and error bars indicate the s.d. Quantification was performed using Zeiss Zen Black and Fiji software⁵⁹.

Intensified 3D arbitrary protein organization at low laser power

For locally precise two-photon excitation in hydrogels, the Ti:Sa Chameleon laser tuned to 800 nm and a Plan-Apochromat 63x/Oil (NA 1.4) objective on the confocal laser-scanning microscope were employed. Photostructuring was conducted in HBS buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.2, 25 °C) supplemented with 50 µM rhodamine B. By the user-defined ROI scanning option in combination with the bleaching mode of the microscope software (Zeiss Zen Black), the diverse shapes and size regions of the portrait of Maria Goeppert-Mayer were precisely written in x/y/z direction. The different ROIs in each z plane were photoactivated for 12 s with an average energy of 3.1 mW (1.2%; max output 2.5 W), a scan speed of 1.54 µs/pixel and a z stack thickness of 5 μm separated by 30 μm. The written structures were visualized by washing with HBS buffer followed by Ni(II)–loading (10 mM NiCl₂ in MQ) and subsequent POI binding via incubation with 300 nM His₆-AF647 in HBS buffer at room temperature. The POI assembly was recorded by CLSM (118 slices z stacks, 96 µm).