Tuning conductivity whilst maintaining mechanical properties in perylene bisimide hydrogels at physiological pH

By using different salts as a method to achieve gelation of two different amino-acid-functionalised perylene bisimides, we were able to tune reduction potentials while maintaining the mechanical and optical properties of the system all at pH 7.4.


Experimental Procedures
All chemicals were purchased from Merck and Alfa Aesar.All chemicals were used as received unless otherwise stated.Distilled water was used throughout.

PBI-H and PBI-L synthesis procedures
Both PBI-L and PBI-H were synthesised by Dr. Draper and have been reported previously. 1,2

Preparation of PBI solutions
Solutions of both PBI-L and PBI-H were prepared at different concentrations from 1 to 10 mg/mL of gelator.For comparative analysis the gels and solutions were prepared at 5 mg/mL of gelator unless otherwise stated.
To prepare a 10 mL solution of PBI-L or PBI-H at 5 mg/mL, 50 mg of the desired PBI is weighed into a vial. 1 molar equivalent of 0.1 M NaOH is then added and then topped up to 10 mL with distilled water.A background electrolyte of 0.1 M NaCl was then added to the solutions (this is for the electrochemical studies, but was in all solutions to ensure there was no effect on UV-vis, rheology etc).The resulting solution is then stirred with a magnetic stirrer overnight, or until the all the solid has visibly dissolved.The solutions were then adjusted to pH 7.4 using 1 µL at a time of 1 M HCl or 1 NaOH.Between each drop the solution was stirred for 2 minutes to allow for equilibration of the pH, until the desired pH is achieved.

Preparation of PBI metal salt gels
Gels using MgCl 2 , CaCl 2 and Ca(NO 3 ) 2 were are prepared in the same way, but using a different container, depending on which method of analysis was being carried out.The metals salts were dissolved in distilled water at a concentration of 200 mg/mL.50 µL of metal salt was then added per mL of gelator solution, so a 2 mL gel would contain 100 µL of metal salt.In each case after the metal salt had been added to the gelator solution it was left sealed 16 hours (overnight) at room temperature (20-25°C) to completely gel before being analysed.This time allows for all gels to be uniformly gelled through the dispersion of the salt throughout the system.From previous studies we know that gelation is complete after this time, and so all gels will be compared with other end-point gels.Any sample that visually did not look homogeneously gelled was not measured as it was assumed that the whole sample had not gelled and would not be an accurate comparison or measurement.
For rheology 2 mL of gels were prepared in 7 mL Sterilin vials. 2 mL of gelator solution was pipetted into the vials.Then 50 µL of metal salt was pipetted onto the top of the solution.Initially a dark spot of gelled material could be seen in the solution, which over time diffuses uniformly to gel the whole contents of the vial.For the aging experiments, gels were prepared in a repeat of 6 in Sterilin vials and left in the lab at room temperature for three weeks without being disturbed.

For UV-vis absorption
This was carried out using a 0.1 mm quartz demountable cuvette, or 1 mm cuvette as stated.The appropriate amount metal salt was pipetted dropwise to cover the surface of the cuvette and then the gelator added on top of this before the top of the cuvette was placed on top (the absolute amounts is determined by the volume of the cuvette, ensuring 0.05:1 mL of salt:gelator).
For SANS 40 µL of metal salt was first added to a 2 mm pathlength quartz cuvette, then 0.8 mL of gelator pipetted on top of the metal salt solution.Again, initially a dark spot at the bottom of the cuvette could be seen which diffuse and gel the whole cuvette.

For Cyclic Voltammetry and EIS measurements
CV was carried out using a custom-made cell, this is to increase the surface area of the working electrode and in order not to disturb the gels.Using this set up we also ensure we were measuring the gel rather than the solution surrounding the gel.The cell was made from two pieces of FTO TEC7 coated glass, with copper tape and a 4 cm diameter o-ring as a 1 mm spacer (see Figure S1 below).This set up uses both FTO as the working and counter as a two-electrode setup.There was no reference electrode (it is plugged into the counter electrode), due to this set up, instead we used the set up to run a ferrocene standard to compare the data to, and ensure the cell was working properly.The background electrolyte was already in the prepared solutions.
To prepare the gels in this set up, the o-ring was placed on the conductive side of one of the pieces of glass.The perylene solution (1 mL) was then pipetted into the o-ring ensuring it was full.Next the metal salt solution was then then added dropwise in regular interval over in the solution, to try and ensure an even diffusion.The second piece of FTO was then placed on top with the conductive side down, ensuring that the solution was in contact with both pieces of glass.The absolute volume was tested with water to ensure the right amount was added.The cell was then sealed with two bulldog clips on the edge of the glass not covered by copper tape and was allowed to gel overnight (16 hours).Uniformity of gelation could be seen by eye, and only uniform gels were measured.Pre-cell culture PBI-L and PBI-H solutions were prepared in H 2 O with a pH of 7.40 at a concentration of 5.0 mg/mL.PBI Solutions were heat sterilized at 121°C.Gels were made using solutions of CaCl 2 , MgCl 2 , and Ca(NO 3 ) 2 at concentration of 200 mg/mL and were filter sterilized prior to use.

Cell viability
For cytotoxicity assays, C2C12 cells were seeded at a density of 40,000 cell/mL in 96/48-well plate and allowed to attach for 24 hours before the addition of PBIs in solution and gel form.After a total incubation period of 20 hours, PBIs were removed, and cultures were rinsed thrice with 1x Dulbecco's phosphate buffered saline DPBS (2662059, Gibco).
For Live/Dead assay, Cytotoxicity was assessed by using Live/Dead TM Viability/Cytotoxicity kit (L3224, ThermoFisher) with Calcein AM and Ethidium homodimer-1 staining live and dead cells respectively.After washing with DPBS, a staining solution composed of 2 µM Calcein and 4 µM Ethidium homodimer-1 was prepared followed by an incubation with cells for 30 minutes at the dark at room temperature.Afterwards, cells were rinsed once with DPBS to remove residual staining solution before visualizing using EVOS M7000 microscope.

Characterisation and analysis methods
pH Measurements pH measurements were performed using a FC200 pH probe (HANNA Instruments) with a 6 mm x 10 mm conical tip.The accuracy of the pH measurements is quoted as ± 0.1.

Rheology
Rheological experiments were only carried out on samples that were stable to inversion.This was used as a screening for the minimum amount of gelator or metal salt required for gelation.Samples were then rheological measured in triplicate to confirm gelation.Yield point quoted at the point at which G′ and G″ deviate from linearity.G′ and G″ were taken from the mid point of the linear viscoelastic region (LVR).
Rheological experiments were performed on an Anton Paar Physica MCR301 or 101 rheometer vane (ST10-4V-8.8/97.5)and cup geometry to minimise loading issues.All measurements were carried out at 25°C.Strain sweeps were performed first in order the frequency sweeps were carried out within the LVR.They were performed at 10 rad/s between 0.1-1000% stain.
Frequency sweeps were performed at 0.5% strain (again within the LVR determined above) between 1-100 rad/s.

SANS
For the SANS, a neutron beam, with a fixed wavelength of 6 Å and divergence of Δλ/λ = 9%, allowed measurements over a large range in Q [Q = 4πsin(θ/2)/λ] of 0.001 to 0.3 Å-1, by using three sample-detector distances of 1.5 m, 8m, and 39 m.The cuvettes were housed in a temperature-controlled sample rack during the measurements.The data were reduced to 1D scattering curves of intensity vs. Q using the facility provided software LAMP.The electronic background was subtracted, the full detector images for all data were normalized and scattering from the empty cell was subtracted.The scattering from D 2 O was also measured and subtracted from the data.The data were normalized to absolute units using a 1 mm thick water sample as secondary calibration standard, with a differential scattering cross section of 0.983 1/cm for the experimental settings used.Last, data were radially averaged to produce the 1D curves for each detector position.Experiment numbers 9-11-1964 and 9-12-598 at the ILL, Grenoble.
The instrument independent data were then fitted to the models discussed in the text using the SasView software package version 3.1.2. 3 PBI-L and PBI-H were fitted using SLD values of 3.024x10 -6 Å -2 and 3.698x10 -6 Å -2 respectively calculated from the NIST website, 4 assuming a density of 1.55 g/cm 3 .

UV-vis absorption spectroscopy
UV-Vis absorption spectroscopy was carried out using an Agilent Technologies Cary 60 UV-Visible spectrophotometer.Absorption measurements were performed 0.1 mm quartz cuvettes.

CV
Cyclic voltammetry was carried out using a PalmSens4 potentiostat (Alvatek Ltd).Voltammograms were measured using 0.1 V/s scan rate.Measurements were collected using PSTrace software (Version 7.2).The samples were prepared as described above using FTO.A background electrolyte of 0.1 M NaCl was used.Blanks of water, electrolyte and metal salt was collected to ensure the reduction potential were not from free metal salt in solution.Measurements were collected in triplicate and at different scans rates, the most representative data sets are shown below at the same scan rate for clarity.

Conductivity measurements
Electrochemical impedance spectroscopy (EIS) was employed for the measurements of the ionic conductivities of the prepared gel samples.The gel samples were sandwiched between two FTO glasses where the contact area and thickness of the gel were 1.2 cm 2 and 0.1 cm, respectively.The EIS data was obtained using a Palmsens4 potentiostat within a frequency range of 50 kHz to 1 Hz and a bias of 0.2 V.The ionic conductivities of the gels were calculated using Equation 1.
Where , d, Rb and S represent the ionic conductivity, the gel thickness, volume resistance and the contact area respectively.The measurements were done in triplicate and the mean values of R b obtained from the circuit fitting which correspond to the intercept of a straight line at high frequency was used to calculate the ionic conductivities. 5,6 Supplementary Data

PBI-L minimum gelation concentration at pH 7.4 Concentration CaCl 2 Amount: 50 μL
MgCl 2 Amount: 50 μL Ca(NO 3 ) 2 Amount: 50 µL 1 mg/mL Frequency (rad s PBI-L fits best to an elliptical cylinder combined with a power law.Using a flexible elliptical cylinder requires the length to be >1038 for a good fit, which is outside the range of the equipment, and so combining an elliptical cylinder with a power law was used.The radius and axis ratio in both cases were very similar, suggesting that either fit is reasonable.The PBI-L gels can all be fitted to the same model.The MgCl 2 gel is different to the other two gels.It is necessary for a greater axis ratio for the best fit.
Constraining the radius and/or axis ratio results in the fit becoming worse as determined visually and by an increase in chi squared.
Table S9.The PBI-H gels and solutions can also be fitted using the same model as for PBI-L In this case, the MgCl 2 triggered gel is very similar to the Ca(NO 3 ) 2 triggered gel.The CaCl 2 triggered gel is however pretty similar.It looks like the gelation leads to a lateral association here as the radius really wants to be double that of the starting PBI-H.

Statistical analysis
For MTT: Ordinary one-way ANOVA for gels

Figure S1 .
Figure S1.Diagram of the eChem set up for measuring CV of the gels.

Figure S8 . 2 ScatteringFigure S9 .
Figure S8.SANS and fit from a PBI-H solution at 5 mg/mL.The fit is shown in red and the collected scattering in black with error bars.PBI-H Ca(NO3)2

Figure S10 .Figure S11 .
Figure S10.SANS and fit from a PBI-H CaCl 2 gel at 5 mg/mL.The fit is shown in red and the collected scattering in black with error bars.PBI-H MgCl2

Figure S17 .
Figure S17.Rheological (a) frequency sweeps at 0.5% strain and (b) strain sweeps at 10 rad/s for PBI-L at 5 mg/mL pH 7.4 with 50 µL of salt after three weeks aging.Performed at 25°C.Filled shapes represent G' and open shapes represent G".Measurements performed in triplicate and error bars calculated from standard deviation.MgCl 2 gels data are purple, CaCl 2 gels data are blue and Ca(NO 3 ) 2 gels data are red.

Figure S19 .
Figure S19.Rheological (a) frequency sweeps at 0.5% strain and (b) strain sweeps at 10 rad/s for PBI-H at 5 mg/mL pH 7.4 with 50 µL of salt after three weeks aging.Performed at 25°C.Filled shapes represent G' and open shapes represent G".Measurements performed in triplicate and error bars calculated from standard deviation.MgCl 2 gels data are purple, CaCl 2 gels data are blue and Ca(NO 3 ) 2 gels data are red.

Figure S20 .
Figure S20.Effect of PBI-L and PBI-H on cell viability using MTT assay.(a) Cells were treated with PBI-L and PBI-H gels at 5 mg/mL using three inorganic salts as gelators, CaCl 2 , MgCl 2 , and Ca(NO 3 ) 2 .(b)Cells treated with PBI-L and PBI-H solutions at concentration of 0.83, 0.45, 0.24, and 0.12 mg/mL.Data was obtained from 8 replicates and analysis was performed based on average of viability ± standard deviation.

Figure S21 .
Figure S21.Live/Dead assay analysis of C2C12 cells treated with PBI-L and PBI-H hydrogels.(a) Representative images of cells labeled with Calcein as live (green) and Ethidium homodimer-1 as red (dead).(b) Quantification of viable cells.C2C12 cells were incubated for 20 hours with or without PBI-L and PBI-H hydrogels at 5 mg/mL gelled with CaCl 2 , MgCl 2 , and Ca(NO 3 ) 2 at 200 mg/mL.Viability represents the percentage of living cells counted at 3 independent positions from each well using Fiji software and analysis was performed based on average viability ± standard deviation.Scale bar is 150 µm.

Figure S22 .
Figure S22.Live/Dead assay analysis of C2C12 cells treated with various concentrations of PBI-L and PBI-H solutions.(a) Representative images of cells labeled with Calcein as live (green) and Ethidium homodimer-1 as red (dead).(b) Quantification of viable cells.C2C12 cells were incubated for 20 hours with or without PBI-L and PBI-H solutions in a dose dependent manner.Viability represents the percentage of living cells counted at 3 independent positions from each well using Fiji software and analysis was performed based on average viability ± standard deviation.Scale bar is 150 µm.

Table S1 .
Minimum gelation concentration for PBI-H.N indicates no gel upon inversion and G indicates a stable gel upon inversion.
2 solution.N indicates no gel upon inversion and G indicates a stable gel upon inversion.

Table S3 .
pH dependence for PBI-H using different amount of CaCl 2 solution.N indicates no gel upon inversion and G indicates a stable gel upon inversion.

Table S4
. pH dependence for PBI-H using different amount of MgCl 2 solution.N indicates no gel upon inversion and G indicates a stable gel upon inversion.

Table S5 .
Minimum gelation concentration for PBI-L.N indicates no gel upon inversion and G indicates a stable gel upon inversion.TableS6.pH dependence for PBI-L using different amount of Ca(NO 3 ) 2 solution.N indicates no gel upon inversion and G indicates a stable gel upon inversion.

Table S7 .
pH dependence for PBI-HLusing different amount of CaCl 2 solution.N indicates no gel upon inversion and G indicates a stable gel upon inversion. PBI-L

Table S8
. pH dependence for PBI-H using different amount of MgCl 2 solution.N indicates no gel upon inversion and G indicates a stable gel upon inversion.PBI-L

pH dependence MgCl 2 Amount 10 μL 30 μL 50 μL
CaCl 2 gel PBI-L Ca(NO 3 ) 2 gel PBI-L MgCl 2 gel Table of SANS fit for PBI-L PBI-L solution PBI-L Figure S7.Rheological strain sweeps performed at 10 rad/s at 25°C for.PBI-H gels formed at 5 mg/mL of gelator with MgCl 2 , CaCl 2 and Ca(NO 3 ) 2 at pH 7.4.Filled shapes represent G' and open shapes represent G".Measurements performed in triplicate and error bars calculated from standard deviation.MgCl 2 gels data are purple, CaCl 2 gels data are blue and Ca(NO 3 ) 2 gels

Table S11 .
Reduction potentials for PBI gels

Table S12 .
Rb and ionic conductivity values for PBI gels