Supporting Information Hierarchical Organization of Organic Dyes and Protein Cages Into Photoactive Crystals

Supporting Information Hierarchical Organization of Organic Dyes and Protein Cages into Photoactive Crystals

Joona Mikkilä, Eduardo Anaya-Plaza, Ville Liljeström, Jose Ruiz-Caston, Tomas Torres, Andrés de la Escosura, Mauri A. Kostiainen

Contents 1. Materials ...... S2 1.1. Apoferritin (aFt) ...... S2 2. Complexation of 1 and 2 ...... S2 2.1 UV–Vis spectroscopy of the 1-2 complex and precursors in PBS buffer ...... S2 2.2 Job plot ...... S2 2.3 Association constant ...... S5 2.4 Fluorescence spectroscopy of the 1-2 complex and precursors in PBS buffer ...... S6 3. Co-crystallization of 1-2 and aFt ...... S7 3.1 Optical microscopy ...... S7 3.2 Disassembly of 1-2-aFt crystals by increasing electrolyte concentration ...... S9 3.3 Small angle X-ray scattering ...... S9 3.4 UV-Vis and fluorescence spectra of the 1-2-aFt crystals ...... S10 3.4.1 Non-washed 1-2-aFt crystals ...... S10 3.4.2 Washed 1-2-aFt crystals ...... S10 3.5 Transmission electron microscopy ...... S11 4. Singlet oxygen generation ...... S11 5. Evaluation of protein stability upon singlet oxygen production ...... S13 6. References ...... S15

1. Materials Octakis(1-methyl-3-pyridiniumoxy)-ZnPc tetrasulfate (1) was synthesized as reported earlier.1 All other reagents, including 1,3,6,8-pyrenetetrasulfonic acid tetrasodium, salt hydrate (2) were commercially available and used without any further purification. The water used in all procedures was Milli-Q purified. Stock solutions of 1 and 2 were prepared by diluting chemicals in pure water, phosphate- buffered saline (PBS buffer, 10 mM Na2HPO4, 2.7 mM KCl, 187 mM NaCl, pH 7.4) or Tris buffer (20 mM Tris, pH 8.5).

1.1. Apoferritin (aFt) Apoferritin (aFt) from Pyrococcus furiosus was ordered from Molirom and it was delivered as a 10 mg/mL water solution. aFt is a 12 nm spherical protein cage with an inner cavity of 8 nm in diameter.2,3 The protein shell consists of 24 protein subunits that each have a molecular weight of 20 kDa. aFt can withstand high pH (8.5–9) and temperature (85–90 °C).

2. Complexation of 1 and 2

2.1 UV–Vis spectroscopy of the 1-2 complex and precursors in PBS buffer UV–Vis spectroscopy of 1, 2 and the 1-2 complex in PBS buffer were recorded using a JASCO V-660 spectrophotometer. Sample solutions (2.4–3 mL) were measured at room temperature using a Hellma QS quartz cuvette with 1 cm light path. Absorbance between 250 nm and 850 nm was scanned with 1 nm resolution.

Figure S1. UV–Vis spectra of 1, 2 and the 1-2 complex in PBS buffer. The ZnPc 1 tend to stack in such conditions. Addition of 2, in turn, increased and shifted the ZnPc Q-band absorption maximum from 669 to 680 nm, until a stoichiometric ratio of 1:1 was reached. Further additions of 2 increased the absorption at 680 nm only slightly.

2.2 Job plot Eleven 3 mL samples with different portions of 1 and 2 in PBS buffer were prepared and their UV–Vis absorption was measured for the job plot experiment (Figure S2). Measurements were carried out using a JASCO V-660 spectrophotometer and a Hellma QS quartz cuvette with a 1 cm light path at room temperature. Absorption spectra were scanned with 1 nm resolution. The concentration of both

components in different samples varied from 10 to 0 µM so that the overall concentration was always 10 µM.

An equimolar job plot diagram was derived from the UV–Vis spectra shown in Figure S2a. ΔA (Aexp - A0) was calculated for each sample, for the job plot diagram shown in Figure S2b, at 631 nm using the following equation

퐴0 = 휀ퟏ푥푐0 + 휀ퟐ(1 − 푥)퐶0 where ε1 and ε2 are the molar extinction coefficients of 1 and 2, 푥 is the molar fraction of 1 and C0 is the total concentration of both components in the solution. -ΔA was presented as a function of the molar fraction of 1, and the highest point occurred at 푥 = 0.5 (Figure S2b), which corresponds to a 1:1 stoichiometry of 1 and 2.

Figure S2. a) UV–Vis spectra of all 1-2 samples measured for the job plot experiment. Samples were prepared in PBS buffer. Molar ratios of 1 and 2 range from 10:0 to 0:10, while the overall concentration of both components stays constant (10 µM) in all samples. b) Job plot diagram derived from measurements presented in (a). ΔA values were calculated according to equation 1 and presented as function of the molar fraction of 1 (푥). According to these data, 1 and 2 self-assemble into a complex with a stoichiometric 1:1 ratio.

2.3 Association constant In order to elucidate the association constant of the 1-2 complex, as well as the self-aggregation constant of 1 a titration was carried out. ZnPc 1 (5 µM) in PBS buffer was titrated with increasing amounts of 2 (from 0 to 10 µM), remaining constant buffer and 1 initial concentrations. Titration was monitored by absorption spectroscopy (Figure S3).

Figure S3. UV–Vis titration of 1 with 2 in PBS buffer.

Association constant of 1-2 complex, as well as self-aggregation constant of 1 in PBS buffer were evaluated by Reactlab™ Equilibria software (Figure S4). Selected optical window from 450 to 850 nm was selected, in order to avoid absorption due to 2. Titration of the Q-band was then adjusted to the following model where the aggregates are assumed to be dimers:

2 2 H ↔ H2; KH-H = [H2] / [H]

H+G ↔ HG; KH-G = [HG] / [H][G] where H, H2 and G stand for ZnPc 1, ZnPc 1 dimer and 2 respectively. KH-H and KH-G correspond to self- association constant and complex formation binding constants, respectively. Values in brackets denotes concentration.

Figure S4. Normalized absorption at 635 (red) and 675 nm (green) and fitted curves (blue and orange, respectively) of 1 (5 µM) titrated with 2.

2.4 Fluorescence spectroscopy of the 1-2 complex and precursors in PBS buffer The fluorescence spectra of 1, 2 and the 1-2 complex in PBS buffer were recorded using a JACSO FP-8600 fluorescence spectrophotometer at room temperature. Samples were excited in a 3.5 mL Hellma QS quartz fluorescence cell, with 669–681 nm excitation wavelength depending on the maximum observed in the excitation spectra measured in advance. Emission was recorded from a 10 nm higher wavelength than the exciting wavelength until 850 nm in 1 nm steps.

Figure S5. Fluorescence titration of 1 with 2 in PBS buffer. Samples were excited at 669–681 nm wavelength depending on the maximum observed in the excitation spectra measured in advance. Fluorescence increased remarkably until a stoichiometric (1:1) ratio of both components was reached. Further added 2 stayed mostly free in the solution.

3. Co-crystallization of 1-2 and aFt 1-2-aFt crystal samples were prepared by combining 5 µL of 2 mM 1 in Tris buffer, 5 µL of 2 mM 2 in water, 5 µL of 0–800 mM NaCl solution and 5 µL of 10 mg/mL aFt in water, in this order and mixing the gained samples gently with a pipet. In the end each sample had 5 mM Tris buffer concentration, while the NaCl concentration varied between 0 and 200 mM. Samples were stored overnight at +7 °C resulting in 1-2-aFt crystals. The stoichiometric ratio of 1/2/aFt in the samples was 100:100:1.

3.1 Optical microscopy Sample solutions (20 µL) were placed on a marienfeld-superior microscope slide (76x26x1.35 mm with a round cavity of 15–18 mm diameter and 0.6–0.8mm depth) and a cover glass was placed on the sample. Samples were imaged with Leica DM4500 P optical microscope equipped with Canon EOS 60D camera using transmission mode. A ruler at the sample position was imaged to calibrate the scale of the images.

Figure S6. Optical microscope images from 1-aFt reference samples and 1-2-aFt samples prepared in 0, 20, 30, 40 and 100 mM NaCl concentrations. Scale bar in all images is 50 µm.

3.2 Disassembly of 1-2-aFt crystals by increasing electrolyte concentration We studied if 1-2-aFt crystals self-assembled in 30 mM NaCl can be disassembled by increasing the NaCl concentration to 100 mM. 20 µL reference sample was taken from the original 40 µL sample solution before increasing the NaCl concentration of the sample. Volumes of sample and reference sample were kept equal by adding equal volumes of 140 mM (2 µL) and 760 mM (2 µL) NaCl solutions to the sample and 30 mM NaCl solution (4 µL) to the reference sample. One night after increasing the electrolyte concentration, the sample was imaged with optical microscope to discover that not a single 1-2-aFt crystal could be found, while there were plenty of them in the reference sample having a constant 30 mM NaCl concentration (Figure S7). This finding showed that the 1-2-aFt crystals self-assembled in low NaCl concentration can be disassembled by increasing the NaCl concentration to 100 mM.

Figure S7. Optical microscope images of a) 1-2-aFt crystals disassembled by increasing the NaCl concentration of the 20 µL sample solution from 30 mM to 100 mM and b) reference sample with constant 30 mM NaCl concentration. In the image of reference sample also some narrow stick-like 1-2 complexes can be observed. Scale bar in both images is 100 µm.

3.3 Small angle X-ray scattering Small angle X-ray scattering measurements were carried out using a rotating anode Bruker Microstar microfocus X-ray source (Cu Kα radiation, λ = 1.54 Å). The X-ray beam was monochromated and focused by a Montel multilayer focusing monochromator (Incoatec). The beam was further collimated by four slits (JJ X-Ray) resulting in a beam size of less than 1 mm at the sample position. The scattering was collected using Hi-Star 2D area detector (Bruker). The used sample-to-detector distance was 1.59 m, and silver behenate standard sample was used to calibrate the q-range. One-dimensional SAXS data were obtained by azimuthally averaging the 2D scattering data. The magnitude of the scattering vector q is given by q = 4π sinθ/λ, where 2θ is the scattering angle. The samples were measured as is. I.e. 10 μL of 1- 2-aFt crystal sample solution was sealed in a metal ring using Kapton tape. Sample thickness was approximately 0.9 mm. The sample environment was evacuated during the measurements to avoid scattering from air.

SAXS measurements from non-powder-like samples with large crystals, (i.e. crystals formed in 30 mM NaCl concentration) were unsuccessful due to the fast sedimentation of large crystals and possible instability of the crystals in the X-ray beam.

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3.4 UV-Vis and fluorescence spectra of the 1-2-aFt crystals 3.4.1 Non-washed 1-2-aFt crystals The UV–Vis spectra of the 1-2-aFt crystals in Tris buffer were recorded using a Biotek Cytation 3 Imaging reader and a Take3 micro-volume plate. Absorbance of 3 µL sample volumes were scanned between 230 and 900 nm with 1 nm increments at room temperature.The fluorescence spectra of the 1-2-aFt crystals in Tris buffer before and after washing were recorded on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) at room temperature. Samples were diluted to a 2 mL volume and measured in disposable PMMA cuvettes (Brand) with four clear faces. Samples were, with 665–679 nm excitation wavelength depending on the maximum observed in the excitation spectra measured in advance. Emission was recorded from a 10 nm higher wavelength than the exciting wavelength until 900 nm in 1 nm steps. 3.4.2 Washed 1-2-aFt crystals 1-2-aFt crystals self-assembled in 30 mM NaCl and reference samples were washed in the following way. 1.5 mL of 30 mM NaCl, 5 mM Tris (pH 8.5) buffer solution was added to the samples and they were centrifuged in a table top centrifuge for 30 seconds, following the removal of supernatants. After the third washing cycle the removed supernatants were colorless, indicating that any excess of free 1 had been removed from the sample solutions. The final volumes of the washed samples were adjusted to 20 µL.

For fluorescence measurements, 7 µL of the washed sample solutions were taken and diluted to 2 mL with 30 mM NaCl, 5 mM Tris (pH 8.5) buffer solution. The fluorescence intensity of the 1-2-aFt sample was over two times higer compared to 1-2 sample when both were excited at 679 nm (Figure S8). This result indicates that 1-2 complexes bind to aFt and the binding is unaffected by the washing. 1-2 forms a thin stick-like complex by itself (Figure S7b) and thus part of it stayed in the sample after washing, yet their emission was lower than that of the actual 1-2-aFt sample. Reference samples of 1 and 2 did not show any fluorescence activity, which was expected, as both of these chemicals are dissolved in the solvent and hence removed in the washing procedure.

Figure S8. Fluorescence measurements of the washed samples where both 1-2-aFt and 1-2 were excited at 679 nm.

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3.5 Transmission electron microscopy 1-2-aFt sample solution in Tris buffer (20 µL) with 20 mM NaCl concentration was prepared and left standing for 10 min. Sample was centrifuged with a table top centrifuge for 30 s and 15 µL of the supernatant was removed. 1 mL of Tris buffer with 20 mM NaCl concentration was added and sample was centrifuged again for 30 s. Supernatant was removed and washing cycle was repeated using water. Final sample volume was adjusted to 20 µL and the pellet was resuspended. 2 µL of the solution was placed on a carbon coated CF300H-Cu grid (Electron Microscopy Sciences) and left standing for 1 min after which the excess solution was blotted away with filter paper. Another 2 µL sample volume was placed on the opposite edge of the grid and dried under ambient conditions for four hours before imaging. Transmission electron microscopy (TEM) images were taken with Tecnai 12 Bio Twin instrument. Several micron sized crystals could be observed (Figure S9).

Figure S9. TEM micrographs of the several micrometer sized 1-2-aFt crystals self-assembled in 10 min.

4. Singlet oxygen generation 1 The singlet oxygen ( O2) quantum yield () of ZnPc 1 was first measured in DMSO following the well- known relative method, based on the photoinduced decomposition of a chemical scavenger (i.e., 1,3- 1 4,5 diphenylisobenzofuran (DPBF) that reacts readily with O2. Non-substituted ZnPc was used as 6,7 reference compound ((DMSO) = 0.67). In detail, the procedure was as follows: 2.5 mL of a stock solution of DPBF (with an absorbance of ca. 1) in DMSO was transferred into a 1 x 1 cm quartz optical cell and bubbled with oxygen for 1 min. A concentrated stock solution of the Pc in the same solvent was then added, in a defined amount to reach a final Q-band absorbance value of about 0.1. The solution was stirred and irradiated for defined time intervals, using a halogen lamp (typically, 300 W). The duration of these intervals are tuned in each experiment, in order to get a decrease in DPBF absorption of about 3–4%. Incident light was filtered through a water filter (6 cm) and an additional filter to remove light under 530 nm (Newport filter FSQ-OG530). The decrease of DPBF concentration with irradiation time was monitored at 414 nm using a JASCO V-660 spectrophotometer and a Hellma QS quartz cuvette with a 1 cm light path. All experiments were performed three times and the obtained data represent

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mean values of those three experiments. The estimated error is  10%. Singlet oxygen quantum yield

() was calculated through the following equation:

푆 푅 푆 푅 푘 퐼푎푇 휙Δ = 휙Δ 푅 푆 푘 퐼푎푇

Where k is the slope of a plot of ln (A0/At) versus irradiation time t, with A0 and At being the absorbance of scavenger at the monitoring wavelength before and after irradiation time t, respectively. IaT is the total amount of light absorbed by the dye. Superscripts R and S indicate reference and sample, respectively. IaT is calculated as a sum of intensities of the absorbed light Ia at wavelengths from the filter cutoff to 800 nm (step 0.5 nm). Ia at given wavelength is calculated using Beer’s law:

−2.3퐴 퐼푎 = 퐼0(1 − 푒 )

Where transmittance of the filter at a given wavelength is denoted as I0 and the absorbance of the dye at this wavelength is denoted as A.

For measuring complex 1-2 and the 1-2-aFt crystals, please see the procedure described in the main text. The main difference with the one described above for 1 in DMSO is that aqueous solubility of the scavenger had to be achieved by dispersing it, from a concentrated solution of DPBF in DMSO (c.a. 1 mM), into a solution of the common neutral pharmaceutical solubilizer Cremophor EL (0.5 % w/w) in Tris buffer, together with the corresponding photosensitizing system (i.e., 1, 1-2 or 1-2-aFt). The limitation of this methodology is that ΦΔ cannot be quantified, but it allows us to estimate and compare qualitatively 1 the efficiency of different PS systems as O2 generators, as described in the main text and shown in Figure 5a and S10. Dark controls were performed, assessing if any of the samples induced DPBF bleaching in the absence of irradiation.

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Figure S10. Plot of the DPBF absorption decay in the Tris buffer with Cremophor EL (0.5 % w/w) for a) 1, b) 1-2, c) 1-2-aFt, d) 1-2-aFt 500 mM NaCl samples.

5. Evaluation of protein stability upon singlet oxygen production Prior to the protein stability studies, we first determined by UV-Vis spectroscopy the irradiation times 1 needed for complete bleaching of the aqueous ADMA scavenger during O2 experiments. This yields an 1 estimation of the irradiation time required to produce an amount of O2 at least 10 times higher than the concentration of the PS system, since the ratio of initial absorbance values between the scavenger and PS was set as 10:1. Based on those date, we decided to irradiate the actual samples for 10 minutes.

1 In order to evaluate the damage of aFt upon photoinduced O2 generation, two highly concentrated 1-2- aFt crystal dispersions were prepared.

Sample 1: c1 = 40 µM, c2 = 40 µM, caFt = 0.2 mg/mL, cNaCl = 0 mM, VFINAL = 2 mL

Sample 2: c1 = 40 µM, c2 = 40 µM, caFt = 0.2 mg/mL, cNaCl = 500 mM, VFINAL = 2 mL Both samples were irradiated for 0, 30, 60, 90, 120, 150, 240 and 600 seconds (Figure S11). After each irradiation period, their UV-Vis spectrum was recorded, and 50 µL aliquots were taken to evaluate the protein stability by SDS-PAGE.

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Figure S11. UV–Vs spectra of samples a) 1 and b) 2 after each irradiation step. 50 µL aliquots were then taken after each step to analyze by SDS-PAGE the stability of aFt in samples c) 1 and d) 2 over irradiation. The gel lines from left to right are the molecular weight marker, native aFt, and the aliquots taken after 0, 30, 60, 90, 120, 150, 240 and 600 seconds of irradiation (from left to right). Molecular weigth markers (MWM) are given on the left (kDa).

For sample 1, a crystal grounding effect was observed by UV-Vis spectroscopy, revealed by the increase in scattering and an increase in the ZnPc Q-band absorption intensity (Figure S9a). This grounding is 1 probably caused by stirring of the dispersion, which is necessary in O2 experiments. For sample 2, in turn, spectroscopic features that are coherent with all components being disassembled are observed (Figure S9b), while no substantial changes in the spectra occur over irradiation. Regarding SDS-PAGE, the 1 aFt protein migrates at the same speed as for the native aFt in all sample lanes, indicating that O2 does not cause degradation of aFt. We can therefore conclude that the presented 1-2-aFt crystals are a highly robust and organized photosensitizing system.

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6. References

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