Visible Light-Enabled Switching of Soft Material Properties Based on Thioindigo Photoswitches

doi:10.21203/rs.3.rs-3220772/v1

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Visible Light-Enabled Switching of Soft Material Properties Based on Thioindigo Photoswitches

https://doi.org/10.21203/rs.3.rs-3220772/v1

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published 14 Dec, 2023

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Thioindigos are visible light responsive photoswitches with excellent spatial control over the conformational change between their trans- and cis- isomers. However, they possess extremely limited solubility in all conventional organic solvents and polymers, hindering their application in soft matter materials. Herein, we introduce a strategy for the covalent insertion of thioindigo units into polymer main chains, enabling thioindigos to function within crosslinked polymeric hydrogels. We overcome their solubility issues by developing a thioindigo bismethacrylate linker able to undergo radical initiated thiol-ene reaction for step-growth polymerization, generating indigo-containing polymers. The optimal wavelength for the reversible trans-/cis- isomerisation of thioindigo was elucidated by constructing a detailed photochemical action plot of their switching efficiencies at a wide range of monochromatic wavelengths. Critically, indigo-containing polymers display significant photoswitching of the materials’ optical and physical properties in organic solvents and water. Furthermore, the photoswitching of thioindigo within crosslinked structures enables visible light induced modulation of the hydrogel stiffness. Both the thioindigo-containing hydrogels and photoswitching processes are non-toxic to cells, thus offering opportunities for new and advanced applications in soft matter materials and biology-related research.

Physical sciences/Materials science/Soft materials/Gels and hydrogels

Physical sciences/Materials science/Soft materials/Polymers

Photoswiches are light absorbing molecules that can be reversibly interconverted between different isomers with distinct chemical and physical properties.^{1, 2} The photo-induced reversible changes in their properties – coupled with the exquisite spatial and temporal light based control – make them an excellent stimuli-responsive platform for a wide range of applications in actuators,³ optics,⁴ molecular motors,^{5, 6} and photobiology.^{5, 7} Within the current toolbox of photoswitches, the most widely used chromophores are derivatives of azobenzenes,^{8, 9} diarylethenes^{10, 11} spiropyrans,^{12, 13} and bisimines.¹⁴ In the biosciences, azobenzene derivatives are often the preferred choice, mainly due to their geometric change induced by trans-/cis- isomerization.^{1, 7, 8} Such molecular motion enables substantial conformational changes in their surrounding environment, allowing biologists to discretely photo-regulate mechanical functions in biological settings for investigation of e.g., cell materials interaction (mechanotransduction),^{15, 16} or the transport of bioactive components across cellular environments.^17–19 However, the spatial control exerted by azobenzenes is limited by a wide distribution of geometries of the cis- isomers, due to the low barriers of rotation around the single C-N bond.²⁰ This limitation is rooted in the main pathway of azobenzene trans-/cis- isomerization, where the conformational change occurs via rotation of the C-N = N-C dihedral torsion angle instead of the inversion of the C-N = N in-plane bending angle.²¹ Such a limitation is also encountered in other photoswitches derived from fulgimides or stilbenes.^{22, 23}

In contrast, thioindigos offer exceptional spatial control over conformational change, enabled by a 180º rotation around the central C = C bond when the molecules interconvert between their trans- and cis- isomers (Fig. 1).²⁴ The absorption of the cis-form is blue-shifted compared to the trans-form, due to the non-bonding interaction between the sulfur and oxygen atoms²⁵ rather than the sterically induced distortion from planarity and impaired conjugation encountered in azobenzene- and stilbene-based photoswitches.^{21, 23} These features make thioindigos excellent candidates for molecular machinery, providing robust spatial control over an attached molecular payload.^{7, 26–28} However, despite the discovery of their photochromism over 100 years ago,^{29, 30} and the extensive exploration of photoisomerization mechanisms,^{24, 25, 31–33} applications of thioindigos in soft matter materials remain rare. The very few reports on thioindigos’ utilisation mostly focused on dye-doping of ferroelectric liquid crystal displays,^{26, 27} solid supports,^34–36 or a molecular tweezers operating at micromolar concentration for the capture and release of metal ions.³⁷

A major limiting factor hindering the application of thioindigos is their “very low solubility in almost all conventional organic solvents and polymers”,³⁸ preventing the covalent incorporation or compatibilization of such entities into polymeric materials. Indeed, the rigid and almost planar structure of both isomers drives the molecules towards aggregation, significantly reducing their mobility and reactivity.²⁴ Thus, suitable side chains such as alkyl and oligomeric siloxane have been introduced – via demanding synthetic procedures – to decrease the aggregation, yet the use of such chromophores is still limited to acting as dopants in a liquid crystal host.^{26, 27, 39} Recent attempts to modify thioindigo scaffolds have resulted in water soluble adducts by virtue of sulfonic acid residues directly attached to the benzyl rings. However, their water solubility is still within micromolar concentration (1–5 µM).^{28, 40} Any higher concentration was reported to result in major solute-solute interaction, and associated aggregation leading to broadening of the UV-Vis absorption spectra, as well as the failure to identify specific isomers in nuclear magnetic resonance (NMR) analysis.²⁸ Therefore, suitable solubilizing strategies are critical to enable the utilization of thioindigos in aqueous environments, paving the way for their applications in chemical and mechanical biology, as well as smart soft matter materials.

Herein, we pioneer a strategy towards the integration of the thioindigo function into polymer main chains, enabling the assessment of the photoswitching of the thioindigo-containing macromolecules in various solvents, including water (Fig. 1). Our approach employs a polymerizable thioindigo linker that can be readily synthesised in the gram scale. The photoswitching of the small molecule is thoroughly investigated, using a tuneable laser system to reveal the optimal wavelength irradiation for photoisomerization via an action plot analysis.⁴¹ Critically, we develop a strategy to incorporate thioindigo into biocompatible crosslinked hydrogel structures, demonstrating – for the first time – that thioindigo photoisomerization can result in the modulation of hydrogels’ stiffness by light in the visible region.

Assessment of photoswitching of a thioindigo bismethacrylate crosslinker

Our approach for the thioindigo linker was designed such that the reagent contains methacrylate function that can participate in conventional free-radical polymerization. Thus, we developed a facile protocol for gram-scale preparation of a bismethacrylate thioindigo (trans-1, Fig. 2a) with an overall yield of 15.4%, and with simple purification procedure that does not require column chromatography (refer to SI, Section 2.1 for the synthesis procedure and characterisation of trans-1). Despite having the methacrylate and ester substitution on the aromatic rings, thioindigo 1 is insoluble in all polar organic solvents including methanol, ethanol, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF). When screening conventional organic solvents, we found that chloroform is the only solvent that dissolves 1 at concentrations up to 5 mM (3 mg·mL^− 1), allowing for UV-Vis absorption and routine NMR analysis. The UV-Vis absorption spectrum of 1 displays a peak at 540 nm, similar to that of the previously reported thioindigos (Fig. 2b). Irradiation of solution 1 at λ_max = 540 nm results in the decrease of the 540 nm absorbance and the appearance of the blue-shifted peak at 490 nm, with an isosbestic point at 503 nm, indicating the conversion to the cis-isomer. Irradiation of the cis-isomer solution by λ_max = 490 nm resulted in the reversion of the absorbance to the trans- species. The UV-Vis absorbances of trans- and cis-thioindigos are better resolved than isomers of azobenzene,⁴² for the reasons explained above.^{24, 32} The reversible trans-/cis-photoswitching can be repeated for more than 10 cycles without any observable changes in the absorbance spectra of both isomers (Fig. 2c), indicating that no significant photodegradation of thioindigo 1 occurs in CHCl₃ by alternating irradiation of green (540 nm) and blue (490 nm) wavelengths.

To determine the optimum wavelength for the photoisomerization that affords the strongest enrichment of each isomer, we employed a nanosecond pulsed, tuneable optical parametric oscillator (OPO) laser coupled with a UV-Vis spectrometer to screen the activation wavelength. By tracking the in situ absorbance measurements during irradiation, we were able to determine the rate of isomerisation, as well as the photostationary cis-/trans-ratio at discrete wavelengths in a photochemical action plot.⁴¹ We start by comparing the wavelength dependent switching efficiency to the absorption spectrum. The action plots of trans- to cis- and cis- to trans-isomerisation are depicted in Fig. 2d as green circles and blue squares, respectively. As one would intuitively expect, the highest switching rate for cis- to trans-isomerisation is observed in the wavelength regions where the absorption of the cis-1 is at a maximum. This is, however, not the case for the trans- to cis-isomerisation, where the fastest switching rate is observed at 500 nm, which is 30 nm blue-shifted from the absorption maximum. Photoinduced switching was found to persist up to λ_max = 600 nm (orange colour), a region where thioindigo 1 shows minimal absorbance. These results alone, however, are not sufficient to inform the best wavelengths for photoisomerization. It is important to also consider the cis-/trans-ratio presented in Fig. 2e. For trans- to cis-isomerisation, the fastest switching rate was observed at 500 nm, yet this wavelength only produces a cis-/trans- ratio of 30% (blue circles, Fig. 2e). A significantly higher ratio of 75% can be achieved with longer wavelengths close to 550 nm (15 nm red-shifted from the absorption maximum), without significant reduction in switching rate. Notably, almost no trans- to cis-isomerization occurred under in the mild ultraviolet regime (350–400 nm). For cis- to trans-isomerisation, high trans-/cis- ratios (green squares, Fig. 2e) are observed in the wavelength region between 440–470 nm, which is blue-shifted relative to the absorption maximum. Taking into account both switching rates and isomer ratios, it was determined that the ideal wavelengths for trans- to cis-isomerisation and cis- to trans-isomerisation are 540–550 nm and 450–470 nm, respectively.

The cis-form of thioindigo 1 was observed to spontaneously revert to trans-isomer at ambient temperature (ca. 24 ºC), with a thermal recovery rate of 4.7·10^− 4 s^− 1 (inset Fig. 2b), similar to those of other previously reported thioindigos in aprotic solvents.^{31, 40} The relatively fast thermal isomerisation prevented us from determining the isomer ratios by ¹H NMR analysis and obtaining a clear spectrum of the cis- isomer, since a 10–20 min interval is required before an NMR spectrum can be recorded post-irradiation. In addition, the concentration required for routine NMR measurements is much higher than that of UV-Vis absorbance measurements (2 mg·mL^− 1 versus 0.2 mg·mL^− 1), leading to a higher thermal cis- to trans- isomerization rate.²⁸ We also observed precipitation during green light irradiation of trans-1 due to the strong aggregation effects of the cis- and trans- isomers in chloroform. Consequently, we were only able to obtain an NMR spectrum of a cis-1/trans-1 ratio of 3/7, by integration of the corresponding aromatic resonances (Fig. S9).

Photoswitching of thioindigo-containing polymer in conventional organic solvents and water

Following the determination of the optimal wavelength for photoisomerization of crosslinker 1, we proceeded to integrate the crosslinker into a polymer structure. Surprisingly, the methacrylate moieties on thioindigo 1 are highly resilient against free radical activation, as no change in ¹H NMR spectrum was observed even when a solution of 1 and azobisisobutyronitrile (AIBN, 1 mol%) in chloroform was heated under reflux at 70 ºC for 48 h. Attempts to use the methacrylate group as an acceptor in Michael thiol addition was also unsuccessful, since conventional base catalysts, including pyridine and triethylamine, did not initiate the thiol-methacrylate reaction; stronger catalysts such as 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) and dimethylphenylphosphine led to the decomposition of thioindigo scaffold and discoloration. The breakthrough reported herein was ultimately achieved by free radical initiated thiol-ene chemistry of the methacrylates of 1 with thiols using AIBN and at 65 ºC in CHCl₃. For polymerization, we selected poly(ethylene glycol) (PEG) as the water-soluble precursor and modified the end-group to confer the thiol function, PEG₄₄-(SH)₂ (M_n = 2176 g·mol^− 1, Ð = 1.03, refer to the SI, Section 2.2 for the synthesis procedure). Thioindigo-containing PEG was subsequently prepared by free radical thiol-ene step-growth polymerization (Fig. 3a), forming polymer P1 with a molar mass of 13,058 g·mol^− 1 (Ð = 2.73). Importantly, P1 is soluble in a wide range of polar solvents, enabling the study of photoswitching by UV-Vis absorbance measurements. Based on our action plot study, we selected the LED light sources with emissions centred at 540 and 470 nm for the reversible photoisomerization of P1 (Fig. S4). Compared to the chloroform solution of small molecule thioindigo bismethacrylate trans-1, P1 displayed similar absorption spectrum in all investigated solvents, and green light (λ_max = 540 nm) irradiation on P1 solutions led to the decrease of the 540 nm peak (Fig. 3b-3e). However, the accompanying absorbances of the cis- isomer are not well resolved, suggesting lower trans- to cis-conversion ratios compared to the photoisomerization of small molecule trans-1.

Inspection of Fig. 3 reveals that the absorbance at 490 nm decreases with increasing solvent dielectric constant after green light irradiation. Specifically, in non-polar aprotic solvents such as dioxane (ε = 2.25) and chloroform (ε = 7.58), the cis-/trans- absorbance ratios at the photostationary state decrease from 0.57 to 0.43, respectively. The cis- to trans-conversion can also be induced by blue light at λ_max = 470 nm, reaching a photostationary with higher trans-isomer content compared to green light treatment. The absorbance spectra in of P1 in polar solvents (those with ε values > 30) indicate complex solute–solute interactions – especially in polar protic solvents such as methanol and water, as can be seen from the broadening of the absorbance (Fig. 3e and 3h). These interactions inevitably limit the assessment of the cis-/trans- ratios at the photostationary states in highly polar solvents. However, the decrease and increase of the 540 nm peak, characteristic of the thioindigo photoisomerization, can be induced by green and blue light irradiation, respectively, suggesting photoswitching of the solutes. Overall, our investigation offers a critical answer for the utility of thioindigo in soft matter materials: thioindigo moieties – when covalently incorporated within polymer main chain – display appreciable photoisomerization in both aprotic and protic polar solvents.

Interestingly, solutions of P1 in water at c ≥ 5 wt% display thermoresponsive gelation behaviour that can be photo-modulated. Specifically, an aqueous solution of P1 at c = 5 wt% formed a solid gel at 37 ºC, reaching a storage modulus of ca. 1000 Pa. Rheological assessment of the hydrogel showed a decrease in the storage modulus to ca. 100 Pa under green light (λ_max = 540 nm) irradiation (Fig. 4a). Blue light (λ_max = 470 nm) irradiation of the mixture resulted in the recovery of the G’ value to approximately 60% of the initial value before green light irradiation. The photoswitching of the G’ value can be induced for several cycles, corresponding to the reversible trans-/cis- isomerization of the thioindigo units. Furthermore, the G’ value can recuperate spontaneously at 37 ºC, to a value similar to those recorded under blue light irradiation (Fig. 4b).

Incorporation of thioindigo into click-crosslinked PEG hydrogels for visible light induced photoswitching of physical properties

Having confirmed the photoswitching of the thioindigos within polymer chains in an aqueous environment, we subsequently designed a strategy to incorporate the photolabile function into hydrogels with robust mechanical strength, enabling photo-modulation of the hydrogels’ stiffness. The very low solubility of the thioindigo bismethacrylate trans-1 prevents direct mixing of the reagent in a resin formulation for crosslinking. Thus, we initially synthesised a polymer precursor P2 from 1 and PEG₁₂-(SH)₂ by free radical thiol-ene polymerization in CHCl₃ (Fig. 5a) The intermediate product P2 has sufficient solubility in DMF at a concentration suitable for polymer crosslinker with a multi-arm linker (> 1 g·mL^− 1). PEG₁₂-(SH)₂ was used in excess, ensuring the remaining thiol function can be employed in the subsequent crosslinking, which occurs via catalyst-free nucleophilic thiol-propiolate addition with a PEG₄₄₈-(propiolate)₄ in DMF (Fig. 5a).⁴³ The resultant organogels were subsequently treated with excess water over 24 h to obtain fully swollen hydrogels. We were thus able to covalently embed various amounts of thioindigo within the network structure, forming a series of hydrogels with increasing weight ratio of thioindigo over PEG component (Fig. 5b), from 0.55 to 5.5 wt%. These materials (Gel_T1 to Gel_T5) allow us to systematically investigate the effect of thioindigo and its photoisomerization on hydrogels’ properties, compared to a PEG-based hydrogel (Gel_T0) with no thioindigo.

We observed a considerable change in the optical and physical properties even with the small amount of thioindigo incorporated into the network structures. Specifically, with 0.55 wt% incorporation of thioindigo, a clear red hydrogel was formed. Increasing the weight content of the thioindigo to ≥ 1.6 wt% resulted in the formation of opaque and deep red hydrogels, as compared to the clear and transparent hydrogel with no thioindigo Gel_T0 (Fig. 5c). Notably, we observed a decrease in the swelling ratio Q_equilibrium (Fig. 5b) and the associated increase in hydrogels’ stiffness (Fig. S11) at the fully swollen state with increasing thioindigo content, despite the decrease in the thiol molar stoichiometry for thiol-propiolate crosslinking (lower crosslinking density in hydrogels with higher thioindigo content). These results indicate the significant physical aggregation of the thioindigo moieties, enhancing the mechanical properties of the networks. The hydrogel with the highest thioindigo content (5.5 wt%, Gel_T5) showed variations in stiffness in response to both temperature change and light irradiation (Fig. 6a). In particular, Gel_T5 displayed an increase in G’ value from 4.1 kPa to 4.7 kPa when the temperature was increased from 24 ºC to 37 ºC. When the hydrogel was subjected to green light irradiation at 37 ºC, the G’ value decreased to ca. 3 kPa, and reverted back to 3.8 kPa under blue light irradiation (Fig. 6b). The photo-modulation of the hydrogel’s stiffness by alternating green and blue light irradiation can be repeated for 3 cycles under rheological assessment, similar to the photo-responsiveness displayed by physically crosslinked hydrogel prepared from P1. Likewise, the green light-induced softened hydrogel slowly recovered its storage modulus in the dark. Other hydrogels Gel_T3 and Gel_T4 also displayed switching of the stiffness, by 3% and 8% variation in G’ value, respectively in response to the changes in green and blue light irradiation (Fig. S11). Hydrogels Gel_T0, Gel_T1 and Gel_T2 did not show any changes in the storage modulus when subjected to light irradiation, and only minor increase in stiffness at elevated temperature (Fig. S11).

Biocompatibility of hydrogels

Biocompatibility is an important parameter for materials intended for cell-related research and biomedical applications. In order to assess whether the hydrogel materials and photoswitching in the current study are biocompatible, Annexin V and Propidium Iodide (PI) staining were performed to evaluate apoptosis of cells exposed to hydrogels and trans-/cis- photoswitching process. Gel_T5, which contains the highest amount of thioindigo and displayed a significant change in stiffness under green light irradiation, was selected for biocompatibility assessments. Figure 7a indicates that HEK-293T cells incubated with hydrogels before and after irradiation with green LED light (λ_max = 540 nm) maintained their polygonal morphology similar to the untreated control. In contrast, HEK-293T cells incubated with the positive control (DMSO) displayed changes in cytoplasmic morphology, appearing as clusters with a rounded shape, suggesting DMSO has a cytotoxic effect on the cells. Crucially, the percentages of live HEK-293T cells treated with hydrogels and the photoirradiation process remained above 90% after 24 h incubation (Fig. 7b-7c). Treatment with hydrogels did not result in significant differences in the percentage of apoptotic cells as compared to the untreated control (Fig. 7c). On the other hand, the cell viability obtained in cells exposed to hydrogels was higher than that of the cells exposed to DMSO.

We further assessed the cytotoxicity of hydrogels using human peripheral blood mononuclear cells (PBMCs) from healthy donors. The majority of PBMCs treated with hydrogels before and after irradiation were alive, achieving a viability of more than 80% (Fig. 7d-7e). As a positive control, treatment with DMSO led to low cell viability, evidence by the high percentage (> 90%) of late apoptotic and necrotic cells (Fig. 7e). Of note, hydrogels before and after irradiation with an LED green light showed similar cell viability in both HEK-293T cells and PBMCs (Fig. 7c and 7e). In summary, these findings indicate that thioindigo-containing hydrogels – and the photoswitching process – both have good biocompatibility with cells.

Thioindios were discovered more than a century ago,³⁰ and their negative photochromism was thoroughly examined in the 1970-1980s.^{25, 32, 37, 38} Despite their advantageous switching properties in the visible wavelength region, applications of thioindigo in (bio)materials to date have been hindered by its strong tendency to aggregate in all organic solvents and water. We address this critical shortcoming by introducing a thioindigo bismethacylate that can: (i) be readily synthesised in gram scale, and (ii) participate in free radical thiol-ene addition for covalent insertion into polymer backbone, via stepgrowth polymerization. Our detailed investigation of the photoisomerization – by mapping the isomers’ conversion as a function of activation wavelength in a photochemical action plot – reveals the optimal wavelength range for maximum enrichment of the cis- or trans-isomers at 540–550 nm and 440–470 nm, respectively. It should be noted that our previous work on action plot investigation has mostly focused on photocycloadditions, photothermal hybrid reactions, and photorelease (Norrish type) reactions, all of which display a red-shift in the photoreactivity compared to their absorption wavelengths.⁴¹ Similarly, we observed a red-shift of the activation wavelength for trans- to cis- isomerization of thioindigo, and an interesting blueshift for cis- to trans-conversion wavelength. These findings provide highly reliable guidance to the irradiation window required for most efficient conversion, compared to the conventional reliance on UV-Vis absorption spectra of the photoactive isomers.

When incorporated into a PEG polymer mainchain, the thioindio units display reversible photoisomerization by green and blue light, similar to that of the monomer compound. This photoswitching behaviour was observed in a range of organic solvents. Interestingly, the self-aggregation of thioindigo moieties induces physical interaction, and the corresponding gelation of thioindigo-containing PEG, in water at physiological temperature (37 ᵒC). The reversible photoisomerization further enables modulation of the hydrogel’s modulus by alternating green and blue light irradiation. Critically, we demonstrated the insertion of the thioindigo units within covalently crosslinker PEG hydrogel, consequently altering the hydrogels optical and physical properties. These hydrogels stiffness can be decreased or increased by green or blue light irradiation, respectively. Our preliminary cell testing on the hydrogels indicates that the thioindigo-containing PEG hydrogels and the photoswitching processes are non-harmful to living cells, and thus our hydrogel scaffolds are suitable for further cell-based study. Altogether, these findings demonstrate the potential of thioindigo photoswitch in polymeric materials, offering well-defined spatial and temporal control over the physical properties. Specifically relating to biology research, the all-visible-light responsive hydrogel platform offers significant potential for personalized disease models, opening new pathway to gain insights into the effects of stiffness changes on disease progression.

Synthetic procedures. Detailed synthetic procedures are described in the Supplementary Information and are accompanied by reaction schemes and NMR characterizations figures.

Photoisomerization Action Plot Measurements. The on-line tracking of absorbance spectra during irradiation was conducted using an in-situ UV-Vis apparatus. An Ocean Optics DH-MINI Deuterium-Tungsten-Halogen lamp was coupled via optic fibres (P400-025-SR) to an Ocean Optics FLAME-T-UV-VIS spectrometer, sensitive from 200 to 850 nm, via a cuvette holder. The custom designed cuvette holder enabled laser irradiation into the bottom of the cuvette, while simultaneously measuring absorbance through the side of the cuvette. An Opolette 355 tuneable OPO, emitting 5 ns pulses from 210–2400 nm at a repetition rate of 20 Hz, was directed upwards into the bottom of a quartz fluorescence (Hellma Analytics quartz high precision cell). The laser energy deposited into the sample was measured above the cuvette holder before and after experiments using a Coherent EnergyMax thermopile sensor (J-25MB-LE) to account for any power fluctuations during irradiation.

Thioindigo trans-1 samples were prepared with a concentration of 48 µmol·L^− 1 in CHCL₃ at 25°C. Kinetic absorbance data at 540 nm was recorded every 250 ms (50 ms integration time, 5 scan average). In additional, full spectra were saved every 10 s during irradiation. All data processing was performed in Matlab®. The slow switching rates at long wavelengths, combined with the competing thermal relaxation, meant that a constant photon flux was not employed for all wavelengths. Instead, the switching rates (measured in s^− 1) were determined using an exponential fit to kinetic absorption measurements and were normalised by the incident photon flux employed at each wavelength (measured in photons s^− 1) to give a normalised switching rate with units of photon^− 1. For full details refer to the Supporting Information section 1.5.

Hydrogels fabrication. In a typical procedure, PEG₁₂-(SH)₂ (7.8 mg, 0.01 mmol, 1 eq. of thiol group) and thioindigo bismethacrylate (0.1–0.5 eq. of methacrylate group, refer to Fig. 5b for the various ratios of thioindigo bismethacrylate used), were dissolved in chloroform (5 mL). AIBN (1 mg, catalytic amount) was added, and the solution was bubbled with nitrogen for 15 min, before being heated at 65 ºC under refluxing conditions for 24 h. Chloroform was evaporated under a gentle flow of nitrogen until a viscous oil was obtained. The residue was dissolved in DMF (0.5 mL), and PEG₄₄₈-(propiolate)₄ (100 mg, 5·10^− 3 mmol, 1 eq. of propiolate group) was added. The solution was subsequently casted onto a poly(dimethylsiloxane) (PDMS) mould to form square shaped organogel within 3 h of curing at 60 ºC. The resultant gel was placed into DI water (500 mL) and allowed to swell over 24 h, with frequent change of water, to obtain thioindigo-containing hydrogel materials.

Rheological studies. Rheological experiments were measured using an Anton Paar Physica rheometer (MCR 302e) with a plate-plate configuration. The lower plate is made of quartz and the upper plate is made of stainless steel with a diameter of 12 mm. A liquid lightguide was placed underneath the lower plate, with other end connected to a Mightex WheeLED™ wavelength-switchable LED sources. In a typical experiment, hydrogel sample (ca. 50 mg) was placed on the quartz plate and the upper plate was brought down to a measurement gap of 0.2 mm. A layer of paraffin oil was applied on the edge of the upper plate to prevent dehydration. The measurement was started by applying a 1% strain with a frequency of 0.1 Hz on the sample.

Cell Cytotoxicity Testing. To evaluate if hydrogels and the photoswitching under green light irradiation have potential cytotoxic effects on living cells, we used the human embryonic kidney (HEK-293T) cell line obtained from the American Type Culture Collection (ATCC, USA). We also used human peripheral blood mononuclear cells (PBMCs) isolated by gradient density technique (Ficoll-Paque plus, Cytiva, USA) from whole blood of individuals with informed consent from the Health Sciences Authority of Singapore. HEK293T cells and PBMCs were cultured in DMEM or RPMI 1640 (ThermoFisher Scientific, USA) respectively, supplemented with 10% heat-inactivated FBS (Biosera, USA), and 1% penicillin/streptomycin (ThermoFisher Scientific) and maintained in a humidified incubator at 37°C with 5% CO₂.

Apoptosis of cells was detected using Annexin V-APC (Biolegend, USA) and Propodium Iodide (PI, ThermoFisher Scientific) staining according to the manufacturer’s instructions. Briefly, HEK293T cells (5·10³ cells/well) or PBMCs (10⁴ cells per well) were pre-seeded in a 24-well plate for 8 h. Subsequently, a piece of hydrogel of about 4.8 mm² hydrogels was placed over the culture in each well. Hydrogels were either non-irradiated or irradiated with an LED green light (λ_max = 540 nm). A negative control (only DMEM or RPMI complete media) and a positive control (complete media containing 20% DMSO) were included to validate the protocol. After 24 h incubation, hydrogels were removed, and microscopic images of cells were taken using an Invitrogen EVOS™ M7000 Imaging System. Subsequently, cells were harvested and washed in cold Annexin V binding buffer, followed by incubation with 100 µL Annexin V binding buffer (BD Pharmingen, USA) containing 5 µL of APC Annexin V and 1 µL of 100 µg/mL PI working solution for 15 min at room temperature in the dark. Then cells were washed with Annexin V binding buffer, and samples were acquired by flow cytometry using the CytoFLEX LX (Beckman Coulter, USA). Thereafter, the data were analysed using the FlowJo software V10 (Three Star, USA).

Contribution

S.L.W. undertook the wavelength-dependent photoisomerization experiments. V.X.T. contributed to the synthesis of small molecules, preparation of the polymers, fabrication and characterisation of hydrogels. P. H. D. N. and M. T.N. L. investigated the biocompatibility of hydrogels and photoswitching processes. X. J. L., C.B.-K. and V.X.T. conceptualised the study. All authors contributed to manuscript preparation and editing.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files. Extra data are available from the corresponding author upon request.

Acknowledgements

C. B.-K. acknowledges funding by the Australian Research Council (ARC) in the context of a Laureate Fellowship underpinning his photochemical research program as well as QUT’s Centre for Materials Science for continued support. S. L. W. acknowledges funding from the Zukunfts Fellowship as part of the Jena Excellence Fellowship Program. P.H.D.N and M. T. N. L. acknowledge the support of Singapore Ministry of Health’s National Medical Research Council under its Open Fund – Individual Research Grant (NMRC/OFIRG/MOH-000643). This research project was additionally supported by National Research Foundation (NRF) Singapore under its NRF Investigatorship Scheme (NRF-NRFI07–2021–0003).

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Visible Light-Enabled Switching of Soft Material Properties Based on Thioindigo Photoswitches

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Visible Light-Enabled Switching of Soft Material Properties Based on Thioindigo Photoswitches

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Introduction

Results

Assessment of photoswitching of a thioindigo bismethacrylate crosslinker

Photoswitching of thioindigo-containing polymer in conventional organic solvents and water

Incorporation of thioindigo into click-crosslinked PEG hydrogels for visible light induced photoswitching of physical properties

Biocompatibility of hydrogels

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