Photocontrolled Drug Release From Donor–Acceptor Stenhouse Adduct Amphiphile Doped Laden Polymeric Hydrogel

ABSTRACT

In chemotherapy, drug dosage should be carefully planned with precise pharmacokinetics. Compared to direct, soluble drug administration, a stimuli‐responsive drug delivery material system provided an on‐demand medication with minimum adverse effects. Among many stimuli, the use of external red light enables manual remote functionality compared to physiological stimuli such as pH. Herein, a 650 nm red‐light‐responsive, metal‐free hydrogel scaffold for stimuli‐responsive drug delivery is reported. The hydrogel network is built by poly(ethylene) glycol thiol and poly(ethylene) glycol acrylate via Michael addition. Donor–acceptor Stenhouse adduct amphiphile (DA), which acts as the moderator of hydrophobic drug doxorubicin, is encapsulated in a polyethylene glycol network to create a drug‐loaded hydrogel scaffold. The preparation of DA is purely aqueous and shows a broad absorbance band over 430–800 nm, which enables potential operation in deep tissue. During the drug release experiments, the presence of amphiphile greatly slows the drug release rate in vitro. By using 650 nm laser irradiation, the drug release from amphiphile‐encapsulated hydrogel can be triggered after the passive diffusion period. The proposed hydrogel serves as an example of the combination of a photo‐switch conjugated amphiphile and a conventional, commercially available polymer hydrogel network, which could serve as a potential smart material for drug delivery.

Donor–acceptor Stenhouse adduct amphiphile acts as a photo‐responsive moderator of model drug doxorubicin, which is encapsulated together in poly(ethylene) glycol covalent hydrogel crosslinked by Michael addition between acrylate and thiol groups. Upon 650 nm red light irradiation, release of DOX was enhanced. The hydrogel matrix showed potential for a photo‐responsive drug delivery system.

1. Introduction

Chemotherapy is a common treatment strategy against cancer using chemical drugs [1]. However, overdose medication would result in a plateau response, which contributes to harmful side effects without further therapeutic effect. To maximize the therapeutic efficacy with balanced and considerable side effects, on‐demand drug release becomes important [2, 3]. Drug delivery systems with favorable pharmacokinetics have been intensively developed to ensure efficacy and safety [4]. In recent decades, drug delivery systems have been designed with stimuli‐responsive functionality, which provides on‐demand drug delivery toggled by external remote stimuli such as light, pH, sound, and magnetic waves [5, 6, 7, 8].

Hydrogel‐based delivery system has been intensively researched for various classes of drugs such as small compounds [9], nucleic acids [10], and antibodies [11]. Innovative designs of hydrogel‐based drug delivery systems could be implanted in tissue and serve as a drug reservoir. Compared to direct administration of the drug, which would diffuse to the circulation system rapidly, the drug could be reserved in the hydrogel implant and ready to be released upon desired stimuli. This minimizes the arrival of the drug to other vital but non‐target organs [12]. In a recent example, a hydrogel fabricated as an injectable supramolecular material could provide potential for delivery of a drug to tissue with minimum invasion due to shear‐thinning property [13, 14]. Furthermore, hydrogels could be designed as a degradable material, ensuring the absence of the hydrogel in the body after therapy [9, 14, 15, 16]. In this work, we focused on the design of a photo‐responsive hydrogel, which could provide on‐demand drug release.

Our recent works focused on the designs of supramolecular assembly of photo‐switch conjugated amphiphile into a macroscopic scaffold [17, 18, 19]. Our reported photo‐responsive amphiphile could self‐assemble into nanostructures such as a tube, ribbon, helix, or sheet in an aqueous system. In the presence of divalent cations, carboxylate hydrophilic tails are stacked via ionic crosslink, turning the nanostructure dispersion into a macroscopic scaffold [20, 21]. The photo‐responsive drug delivery application potential of such a system has been demonstrated in a stiff‐stilbene amphiphile using hepatocellular carcinoma cell line PLC/PRF/5 with multikinase inhibitor drug Sorafenib in vitro [22]. This suggested that a photo‐switch conjugated amphiphile could act as a potential drug moderator.

Donor–acceptor Stenhouse adduct (DASA) is a recently invented photo‐switch with a visible light absorption spectrum [23, 24]. We previously reported the donor–acceptor Stenhouse adduct amphiphile (DA), which could self‐assemble into nanofibers in aqueous solution. Upon white light irradiation, the nanofibers convert to nano‐vesicles due to the photo‐isomerization transformation at the molecular level. White light‐controlled release of Rhodamine B and Rhodamine 123 from DA supramolecular scaffold was demonstrated previously [25]. Designs of DASA‐based material systems for biomedical applications have been reported [26, 27]. Our material system based on aqueous DA thus enable to minimize involvements of organic solvents during material fabrication. Furthermore, DA exhibited a broad absorption band over the visible‐light region in aqueous media (430–800 nm) compared to organic conditions (450–685 nm), thus favoring a red‐light‐controlled material system for potential operations in deep tissue. However, macroscopic soft scaffolds fabrication of DA supramolecular assemblies requires the cross‐linking of a divalent cation, which might induce sensitivity and reduced biocompatibility toward live cells [25]. Herein, we encapsulated the DA in a polymer hydrogel system, which was crosslinked by Michael Addition between poly(ethylene) glycol thiol (PEG‐thiol) and poly(ethylene) glycol acrylate (PEG‐acrylate). Together, the scaffold functions as a geometry‐standardized drug delivery material with red light responsiveness. In this work, Doxorubicin (DOX) was used as a model drug. Notably, DOX is a common hydrophobic anti‐tumor chemotherapeutic with various side effects in humans, such as cardiotoxicity [28, 29, 30]. Previous studies reported the self‐assembly system, such as micelles, for the delivery of DOX [31, 32, 33]. We speculated that the DOX could bind to the hydrophobic section and the fully conjugated system of DA. DOX would dissociate from the DA nanostructure upon photo‐isomerization from open‐DA to cyclized‐DA. We aim to develop a hydrogel system with an on‐demand drug release function to balance the therapeutic effect and harmful side effects, using DOX as an example. Furthermore, DOX also showed fluorescence properties with excitation around 480 nm and an emission range of 550–650 nm, thus allowing us to monitor the drug release of DOX in the smart hydrogel system [34]. In this work, we demonstrated that the photo‐switch conjugated amphiphile, DA, acted as a photo‐responsive drug moderator when encapsulated in a commercially available polymeric hydrogel, which provides a new smart drug delivery system controlled by red light (Scheme 1; Figure S1).

SCHEME 1.

Schematic illustration of DOX‐loaded hydrogel preparation. For DOX‐loaded DA‐encapsulated hydrogel, 5 µL DA dispersion (concentration varied in different groups according to methodology) was mixed with 5 µL DOX (1 mM), PEG‐thiol (10 wt.%) and PEG‐acrylate (10 wt.%). For the DOX‐loaded control hydrogel, MQH2O was used instead of DA dispersion. After incubation in a circular disk mold, a hydrogel was formed by Michael Addition crosslink.

2. Results

2.1. Photo‐Isomerization of DA Induced by 650 nm Irradiation

Previous work has reported the photo‐isomerization of the DA in both organic and aqueous environments induced by white light [25]. Herein, we tested whether the 650 nm laser irradiation can induce the photo‐isomerization of DA. We use 1 equivalent of NaOH to prepare 86 µM DA in aqueous solution. Consistent with previous studies, aqueous solution of DA showed two absorption bands over 250–325 nm and 430–800 nm, respectively. Upon 650 nm irradiation, the absorption band over 250–325 nm increased, and the absorption band over 430–800 nm decreased, with identified isosbestic points at 279 and 454 nm (Figure 1a), suggesting selective photo‐isomerization from open‐DA to cyclized‐DA. When monitoring the absorbance at 650 nm, the absorbance remained unchanged in the first 15 min non‐irradiation period. After switching on the irradiations, the absorbance at 682 nm kept decreasing (Figure 1b). This suggests the 650 nm laser irradiation is adequate to induce conformational change of DA as in a previous study. This suggests the 650 nm laser irradiation is adequate to induce conformational change of DA, though it might be partially induced by local heat upon photoirradiation. Notably, the broad absorption band of DA over 430–800 nm is attributed to polarity effect and self‐assembly in aqueous solution. The gradual change of the absorption band to a narrowed range of 450–685 nm by increasing Tetrahydrofuran/water solvent ratio was reported previously [25]. We consider that the use of red light is a significant advantage in drug delivery design since red light (600–700 nm) has a lower absorption coefficient in skin tissue across the visible light spectrum, therefore favoring the application of external stimuli triggered drug release in deep tissue [35].

FIGURE 1.

Photo‐isomerization of DA induced by 650 nm irradiation in aqueous media. (a) UV–vis absorption spectra of 86 µM DA in aqueous media. 650 nm light irradiation was turned on after 15 min. Black line: Before irradiation (0 min); Orange line: upon irradiation (20–70 min); Red line: After 60 min irradiation period (75 min). The arrows indicate the change of the absorption bands over 235–350 nm and 430–800 nm, respectively. (b) Time‐course of the 650 nm irradiation of aqueous DA (86 µM) monitored at 682 nm (Red line) over 75 min.

2.2. Biocompatibility of Aqueous DA

Biocompatibility is a key concern in drug delivery material design. Our DA could act as a cell‐material interface for live cell culture in a macroscopic scaffold format as reported previously [36]. Meanwhile, we attempted to supplement the aqueous DA into the cell culture medium for the biocompatibility assay. Using HeLa cells as a model, we found that the 100 µM DA caused around a 15% drop in cell viability on average (Figure 2a). We also conducted a cell viability test using human mesenchymal stem (hMSCs) cells as a normal cell model. Around a 24% cell viability drop upon hMSCs culture for 3 days with 100 µM DA (Figure S2). We also stained both live cells and dead cells in HeLa cell culture supplemented with 100 µM DA. Well‐spread HeLa cells with a green calcein signal were observed. A few red dots indicated dead cells (Figure 2b). These observations suggested HeLa cells remain healthy in culture with DA supplementation, similar to the DA‐free control group. We speculate that DA is a potentially safe organic compound when acting as a drug moderator in the drug delivery system.

FIGURE 2.

Biocompatibility of aqueous DA in HeLa cells. (a) MTS experiment for cell viability of HeLa cells cultured with DA of different concentrations (20–100 µM) after 3 days of culture. (b) Live/Dead staining of HeLa cells after 2 days of culture with or without 100 µM DA. Green: Calcein AM (Live stain); Red: EthD‐1 (Dead stain); scale bar: 500 µm (for all images).

2.3. Fabrication of DA‐Laden PEG Polymer Hydrogel

To encapsulate the DA in a polyethylene glycol (PEG) network, aqueous DA was mixed with both PEG‐thiol and PEG‐acrylate in Dulbecco's phosphate‐buffered saline (PBS), which were subsequently crosslinked via Michael addition [37]. Noted that a higher concentration of DA is used in the preparation of hydrogel compared to the biocompatibility test. In scanning electron microscopy (SEM), both the PEG hydrogel and the DA‐encapsulated PEG hydrogel (DA/PEG hydrogel) showed a porous surface. At higher magnification, both hydrogels exhibited a rough surface (Figure 3). Fourier‐transform Raman spectroscopy was conducted to verify the successful crosslinking via Michael addition and encapsulation of DA. C═C functional group is featured in the Raman shift 1420 cm⁻¹ in PEG‐acrylate solution but not in PEG‐thiol solution and PEG hydrogel. The signal disappeared after the formation of hydrogel, thus suggesting the transformation of the alkene group upon Michael addition (Figure S3a). Aqueous DA is featured with signal peaks in the range of 1000–2000 cm⁻¹, while PEG hydrogel is featured with signal peaks in the range of 2700–3000 cm⁻¹. Upon encapsulation, the two feature signal bands remained in the DA/PEG hydrogel, thus suggesting the successful encapsulation of DA in PEG hydrogel (Figure S3b).

FIGURE 3.

Scanning Electron Microscopy of DA encapsulated PEG hydrogel (DA/PEG hydrogel) and PEG hydrogel without loading DOX. Scale Bar: 500 µm (Low magnification) and 10 µm (High magnification).

2.4. Passive Release of DOX From DA Laden PEG Polymer Hydrogel

To load the model drug DOX in the scaffold, we mixed DOX with the DA and hydrogel precursor solution during scaffold fabrication (denoted as DA+DOX/PEG). For the DA‐free control hydrogel, DA was replaced by water (Figure S1). DOX is a well‐known chemotherapeutic for cancer [28]; the toxicity has been confirmed in HeLa cells (Table S1). By observation, the cylinder‐shaped hydrogel products appeared dark blue color, while the PEG hydrogel appeared transparent (Figure S4). We investigated the drug release of DA+DOX/PEG hydrogel and the control hydrogel (DOX/PEG) in PBS incubation without any stimuli. On day 7, the release of DOX from DOX/PEG was more than 80%. In contrast, around 72% of DOX was released from DA+DOX/PEG hydrogel (226 µM DA). When more DA was encapsulated (567 µM and 1.13 mM DA), the drug release on day 7 was reduced to 54% and 39%, respectively (Figure 4a; Figure S5). This implied the retention of DOX attributed to the encapsulated DA. This is possibly attributed to the low porosity in the DA/PEG scaffold compared to the PEG control scaffold. The presence of porosity in the PEG control scaffold might allow the immediate release of DOX within 1 day. Besides, DA provides higher binding affinity to DOX due to its amphiphilic feature and fully conjugated system, which the hydrophilic section of DA provides enhanced binding affinity to the highly polar amine group and carboxyl group of DOX, i.e., amine group and carboxylic acid group; and the hydrophobic interaction between DA and DOX [31, 32, 33]. The drug releases were similar between day 3 and 7, which suggested that no further drugs were released in this period for both DA+DOX/PEG hydrogel and DOX/PEG hydrogel. After incubation in PBS for 7 days, the hydrogels (567 µM DA) swelled by no more than 20% in diameter on average (Figure 4b).

FIGURE 4.

Passive release profile (Diffusion) of DOX from DA+DOX/PEG hydrogel (567 µM DA) or DOX/PEG hydrogel. (a) DOX release profile by diffusion from DA+DOX/PEG hydrogel (567 µM DA) or DOX/PEG hydrogel to surrounding PBS over 7 days, n = 4 hydrogels. (b) Diameter of hydrogels (average ± standard deviation) on day 7, n = 4 hydrogels.

2.5. 650 nm Irradiation‐Induced DOX Release From DA Laden PEG Polymer Hydrogel

For the light‐controlled drug release experiment, where the summary of the experiment parameters was provided (Table S2), we allowed 2 days of passive release of DOX from hydrogels so the subsequent change in drug release would be attributed to the 650 nm irradiation treatments with minimized diffusion effect of initially unconjugated DOX (Figure 5a). Similar to the passive drug release experiment, the PEG‐only control hydrogel showed more DOX release (80%) compared to DA encapsulate hydrogel (40%) in the first two days (Figure 5b). We applied 650 nm light irradiation vertically to the hydrogel; a rectangular, transparent region was created (Figure S6). The complete transparency was observed in irradiated hydrogel but not in aqueous DA during UV‐spectroscopy analysis, attributed to differences in concentration, optical path, and duration time. Notably, the optical path in UV spectra analysis was 1 cm while the thickness of the hydrogel, by calculation, is 1.075 mm. This suggested the photo‐isomerization of DA when encapsulated in a polymeric hydrogel. For each hydrogel, we performed 15 min irradiation treatment thrice. We performed 650 nm irradiation treatments on either day 2 or 7 (Figure 5a). Before light irradiation treatment, PBS was refreshed. For the day 2 treatment group (Day 2 light DA), more DOX (25%) was released after incubation until day 7, compared to the control group (21%, p = 0.00249) (Figure 5c). Similarly, for the day 7 treatment group (Day 7 light DA), more DOX (13%) was released after incubation until day 7, compared to the control group (9%, p = 0.00055) (Figure 5d). DA encapsulated hydrogels (Day 2 light DA) showed similar drug release (7.99%) on day 9 as compared to irradiation‐free DA encapsulated hydrogels (8.60%). This suggested that the light treatment boosted the drug release from the hydrogels. After 5 days period for the drug release from the irradiated region and subsequent replacement of the surrounding medium, the previously irradiated hydrogels followed similar drug release as hydrogels that were not irradiated. Meanwhile, for all DOX/PEG hydrogels, DOX release profiles remained similar regardless of being treated with 650 nm irradiation. This might be attributed to either instant diffusion of DOX during the first 2 days or absence of photo‐responsiveness at 650 nm irradiation. Notably, there was around 80% DOX loss in the control hydrogel, which suggested that only 20% DOX was available in the subsequent light irradiation‐mediated drug release experiment (Figure 5a). We investigated a hydrogel with double the amount of DA (1.08 mM) and DOX (10 nmol) without any passive diffusion period and prolonged incubation. However, the releases were 13.5% and 10.4% (p = 0.02154) difference between light DA and no light DA, respectively. This might be considered statistically insignificant (Figure S7). We speculate that the initial passive period, prolonged incubation, and the concentration of drug and moderator would be important in our photo‐switch conjugated amphiphile laden polymeric hydrogel drug delivery system. Besides, we also investigated a lower irradiation time. We compared 3 min irradiation and 15 min irradiation in each region (each hydrogel received three irradiations) and identified that the drug releases of the two groups are similar, and both groups showed more drug release compared to the irradiation‐free control group at 53 h. (Figure S8). This suggested that 3 min irradiation per region is sufficient to trigger drug release from the hydrogel.

FIGURE 5.

DOX release profile from DA+DOX/PEG hydrogel (denoted as DA) and DOX/PEG hydrogel (denoted as PEG) before and after 650 nm irradiation. (a) Schematic illustration representing the schedule of the light‐controlled drug release experiment. (b) Passive release profile from 0 h to 2 days. (c) On day 2, PBS was refreshed for all groups; “Day 2 light DA” and “Day 2 light PEG” received 650 nm irradiation treatments and were incubated until day 7. ^* indicated that the p‐value of a two‐tailed t‐test analysis (unequal variance) was lower than 0.003, where “Day 2 light DA” and “No light DA” were compared on day 7. (d) On day 7, PBS was refreshed for all groups; “Day 7 light DA” and “Day 7 light PEG” received 650 nm irradiation treatments and were incubated until day 9. # indicated that the p‐value of a two‐tailed t‐test analysis (unequal variance) was lower than 0.003, where “Day 7 light DA” and “No light DA” were compared on day 9. Light = 7 hydrogels for each condition; no light = 14 hydrogels for each condition.

Based on the calculation, the DA encapsulated gel showed minor swelling after 7 days (from 15 to 16.125 mm in diameter, Figure 4b). The approximated area of three irradiation rectangular regions is 7.5 mm²; the approximated area of the hydrogel is 204.2 mm². Therefore, the irradiated area is about 11% of the hydrogel's area. Considering only 60% of the drug remains after the initial passive diffusion period, around 6.6% drug release is expected. 4.12% increment (day 2 light vs. no light on day 7) and 4.39% increment (day 7 light vs. no light on day 9) might be attributed to continued passive release of drug in non‐irradiated, DA encapsulated hydrogel. 4% DOX (0.2 nmol) release would contribute to 0.0667 µM DOX in the surrounding medium. The cytotoxicity of DOX over the micromolar scale was investigated (Table S1). Besides, as DOX binds to DA by electrostatic interaction between the charged amine (NH₃ ⁺) of DOX and the deprotonated carboxylate (COO⁻) on the DA. Irradiated aqueous DA showed a change in nano‐structure from nano‐fiber to nano‐vesicle. The release of DOX from a polymeric hydrogel based on the release of nano‐vesicles.

Cell viability of PEG hydrogel and DA+DOX/PEG hydrogel was tested using HeLa cells and hMSCs. HeLa cells and hMSCs showed 99% and 95% cell viability in the PEG hydrogel group, respectively. We considered that PEG hydrogel is biocompatible. HeLa cells and hMSCs showed 43% and 70% cell viability in the DA+DOX/PEG hydrogel group, respectively (Figure S9). The reduced cell viability might be attributed to the passive diffusion of DOX. To conclude, we reported a metal‐free, organic solvent‐free preparation method to encapsulate aqueous DA in a polymeric hydrogel. The simple manufacture, direct mixture, and subsequent incubation would favor biomedical applications such as drug release. Compared to previous work, using a polymeric hydrogel could standardize the geometry of the scaffold. Addition of DA in polymeric hydrogel could prevent the instant release of hydrophobic drug DOX and provide a red‐light remote‐control functionality for on‐demand DOX release from hydrogel drug reservoir. The current photo‐responsive amphiphile DA can act as a photo‐responsive drug moderator in a covalent polymer hydrogel, providing an alternative material strategy for precision and controlled drug delivery.

3. Conclusion

To summarize, DA could respond to 650 nm irradiation in aqueous form and show good biocompatibility. In covalent PEG hydrogel, DA acted as a photo‐responsive moderator for DOX release. It prevented the instant, complete passive release of DOX and enabled the 650 nm‐irradiation remote control function for on‐demand release. Compared to the macroscopic scaffold format reported previously, the use of a covalent hydrogel might standardize the geometry of the scaffold and the dose of the drug, which are crucial for biomedical applications. Future directions would include exploring the drug release ability of other photo‐responsive amphiphiles or applying the scaffolds in an animal chemotherapy model.

4. Experimental Section/Methods

4.1. Donor–Acceptor Stenhouse Adduct Amphiphile

DA, molecular weight: 580.3033, was synthesized and characterized in a previous report [25].

4.2. UV–vis Light Spectrum and Raman Spectroscopy Analysis

DA was dissolved using 1.0 molar equivalent NaOH for all preparations in this study. The UV–vis spectra of DA (86 µM) in a 0.2 cm path length quartz cuvette upon 650 nm laser irradiation (0.2 W) were conducted using an Agilent Cary 60 UV–vis Spectrophotometer. Measurements were performed every 5 min. Laser irradiation was turned on at 15 min. The measurements ended at 75 min. Raman Spectroscopy of (1) PEG acrylate (10 wt.%), (2) PEG thiol (10 wt.%), (3) aqueous DA (567 µM), (4) PEG hydrogel (9.47 wt.% PEG) and (5) DA encapsulated PEG hydrogel (567 µM DA, 9.47 wt.% PEG) were recorded using Renishaw Micro‐Raman Spectroscopy using 785 nm laser source and 532 laser source.

4.3. Cell Culture and Biocompatibility Test

HeLa cells were cultured using Dulbecco's Modified Eagle Medium (GibcoTM, Thermo Fisher:11965092), supplemented with 10% Fetal Bovine Serum (Gibco, Thermo Fisher: A5209402) and 1% Antibiotic–Antimycotic (100X) (Gibco, Thermo Fisher:15240062) under 37°C, 5% CO₂. For the quantitative biocompatibility test, 96‐well plates were used. 5,000 HeLa cells were seeded in a 96‐well plate. Aqueous DA was supplemented to the medium, yielding the final concentration of DA: 20, 40, 60, 80, and 100 µM (number of tests = 4; n = 4 wells per test). After 3 days of culture, CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) was diluted in medium at a 1:5 volume ratio and replaced the DA‐supplemented medium. After 1 h incubation, the absorbance at 492 nm was measured using Labexim Products LEDETECT 96 Microplate Reader (Reference filter 620 nm). The toxicity of DOX, ranging from 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, and 4.0 µM, was investigated similarly (number of tests = 2; n = 4 wells). We used one set, n = 4, well for cell‐free negative control (MTS solution + medium) in MTS tests. For Live/Dead staining, 2‐well slides were used. 50 000 HeLa cells were cultured using 2 mL medium with (or without) supplemented with 100 µM DA. After 2 days of incubation, live and dead cells were labelled using 0.5 µM Calcein AM (Invitrogen, Thermo Fisher: C3100MP) and 0.5 µM Ethidium Homodimer‐1 (Invitrogen, thermofisher: E1169), respectively. Images were taken using a Leica TCS SPE Confocal Microscope. Calcein AM: Ex = 488 nm, Em = 500–531 nm; Ethidium Homodimer‐1: Ex 561 nm, Em = 600–632 nm.

hMSCs (Lonza, PT‐2501) of no more than passage 6 were used in this study. Before the biocompatibility test, hMSCs were expanded using MEM α, nucleosides (Gibco, Thermo Fisher: 12571‐063) containing 10% Fetal Bovine Serum (A5209402, Gibco, Thermo Fisher: A5209402) and 1% Antibiotic‐Antimycotic (Gibco, Thermo Fisher:15240‐062). 10 ng/mL of basic fibroblast growth factor (Sino Biological: 10014‐HNAE) was supplemented during cell expansion.

Lactate dehydrogenase (LDH) release assay (thermofisher: C20300) was used in biocompatibility experiments. For cytotoxicity of aqueous DA on hMSCs, 2000 hMSCs were seeded in each 96‐well, and incubated with ranged concentration of aqueous DA: 20, 40, 60, 80, and 100 µM (n = 3). After 3 days of incubation, hMSCs were washed with PBS. The remaining cells were then lysed for the LDH release assay. The absorbance at 492 nm due to formazan was measured using Labexim Products LEDETECT 96 Microplate Reader (Reference filter 620 nm). Higher absorbance refers to more remaining cells, therefore lower cytotoxicity.

For cytotoxicity of hydrogels experiments, 6‐well plates equipped with transwell inserts (Corning: 3460) were used to separate cells and hydrogel. Before the experiment, 50 000 hMSCs were seeded in each well overnight. (1) PEG hydrogel without DOX and DA; (2) PEG hydrogel containing 567 µM DA and 5 nmol DOX was fabricated and incubated in PBS for 2 days and washed, allowing removal of mobile DOX. After that, hydrogels were put on the upper well to prevent mechanical damage to cells. After 3 days of culture, the medium was replaced with 1% Triton X‐100, followed by a subsequent 30 min incubation, to quantify the cell viability using an LDH release assay. The cytotoxicity experiment was also conducted using HeLa cells (n = 3) under the same conditions as hMSCs transwell culture. In the LDH release assay, phenol red‐free medium (HeLa cells: DMEM, Thermo Fisher: 21063‐029; hMSCs: MEM α, Thermo Fisher: 41061‐029) was used and supplemented with 2% FBS and 1% anti‐anti.

4.4. Fabrication of DA‐Encapsulated Hydrogel and Scanning Electron Microscope Imaging

PEG hydrogel was used. Both 4‐arm‐PEG‐thiol (molecular weight: 10 000) and 4‐arm‐PEG‐acrylate (molecular weight: 10 000) were purchased from Jenkem Technology. PBS was used to dissolve the PEG‐thiol and PEG‐acrylate. To encapsulate DA in the hydrogel, we mix DA aqueous suspension (21.5 mM, 5 µL), 5 µL PBS, PEG‐thiol (10 wt.%, 90 µL) and PEG‐acrylate (10 wt.%, 90 µL). The mixture was added to the central region of the confocal dish (15 mm diameter), which served as a mold. The mixture was incubated until solidified; hydrogel was formed by crosslinking between thiol and acrylate (Michael addition). Control hydrogel (without DA) was prepared using 5 µL water instead of 5 µL DA suspension. To prepare scanning electron microscopy samples, the hydrogels were incubated in water and freeze‐dried to obtain white color scaffold and blue color scaffold, which respectively represent the control hydrogel and the DA‐encapsulated hydrogel. After that, the scaffolds were placed on conductive adhesive carbon tape. Scanning electron microscopy was performed using a Tescan VEGA3 scanning electron microscope.

4.5. Passive Release of DOX From DA‐Encapsulated Polymer Hydrogel

Hydrogels of three different DA concentrations (226 µM, 567 µM, and 1.13 mM) were compared for the passive drug release profile. Doxorubicin (DOX) was loaded into the hydrogel by mixing DOX and hydrogel precursor solution. Each hydrogel (190 µL) contained 5 nmol DOX. The hydrogels were put in a 6‐well containing 5 mL PBS for passive release of DOX. Measurements (226 µM DA: 2 h, 3d, and 7d; 567 µM DA: 0 h, 2 h, 1d, 3d, and 7d; 1.13 mM DA: 2 h, 1d, 3d, and 7d) were done using a Thermo Scientific Varioskan LUX Multimode Microplate Reader. We used equation y = 1.6747x + 0.1722 for fluorescence (a.u.) data of 0–1.7543, and y = 1.8183x + 0.4029 for fluorescence (a.u.) data of 1.7543–7.4193 (Figure S10). Meanwhile, the diameter of the hydrogels was also recorded.

4.6. Red Light‐Triggered Release of DOX From DA‐Encapsulated Polymer Hydrogel

PEG hydrogel with 567 µM DA was used to investigate the DOX release upon red light stimuli. Three control groups were performed: (1) DA encapsulated hydrogel without light stimulation; (2) Control hydrogel with light stimulation; (3) Control hydrogel without light stimulation. The hydrogels were immersed in a 12‐well plate with 3 mL PBS. After around 2 days of passive release, a 650 nm red light laser was set vertically to irradiate the hydrogel. Hydrogels were irradiated on either day 2 or 7. Each hydrogel received three 15‐min irradiation treatments. The drug release responses were monitored up to 9 days. Lowered irradiation time (3 min each region) was also investigated. The drug release after replacement of PBS on day 2 was monitored and ended on day 4.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: marc70360‐sup‐0001‐SuppMat.pdf.

Wong W.‐K., Leung M.‐H., Hung K.‐L., et al. “Photocontrolled Drug Release From Donor–Acceptor Stenhouse Adduct Amphiphile Doped Laden Polymeric Hydrogel.” Macromolecular Rapid Communications 47, no. 16 (2026): e70360. 10.1002/marc.70360

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.