Ruthenium-Crosslinked Hydrogels with Rapid, Visible-Light Degradation

Abstract

Incorporation of photoresponsive molecules within soft materials can provide spatiotemporal control over bulk properties, and address challenges in targeted delivery and mechanical variability. However, the kinetics of in situ photochemical reactions are often slow and typically employ ultraviolet wavelengths. Here, we present a novel photoactive crosslinker Ru(bipyridine)₂(3-pyridinaldehyde)₂ (RuAldehyde), which was reacted with hydrazide-functionalized hyaluronic acid to form hydrogels capable of encapsulating protein cargo. Visible light irradiation (400 – 500 nm) initiated rapid ligand exchange on the ruthenium center, which degraded the hydrogel within seconds to minutes, depending on gel thickness. An exemplar enzyme cargo, TEM1 beta-lactamase, was loaded into and photoreleased from the Ru-hydrogel. To expand their applications, Ru-hydrogels were also processed into microgels using a microfluidic platform.

Graphical abstract

A ruthenium-containing crosslinker, [Ru(bpy)₂(3-pyridinaldehyde)₂] was synthesized for reaction with hydrazide-modified hyaluronic acidto form visible-light-responsive macro- or microgels. Ru-hydrogel was formulated to encapsulate active protein until photorelease. This new RuAldehyde crosslinker promotes efficient photodegradation while minimizing toxicity.

Dynamic, stimuli-responsive materials can be designed to provide spatiotemporal control over useful physicochemical properties. Responsive polymeric materials have biomedical applications including therapeutic delivery of payloads,^[1–4] control of cell fate,^[5] and dynamic materials that facilitate tissue regeneration.^[6] Hydrogels are particularly useful biomaterials due to their tuneable properties^[7] and programmable response to various cues,^[8] including pH,^[9] local enzymatic activity,^[10] ultrasound,^[11] magnetic fields,^[12] heat,^[13] and light.^[14] Hydrogels are attractive vehicles in which to encapsulate and release proteins, which are typically denatured if exposed to non-aqueous environments or interfaces.^[7] Hydrogel formulations avoid harsh emulsion steps and incorporate high water content compared to polymeric^[15] and lipid-based^[16] materials, preserving proteins in their native state.

Photoresponsive hydrogels offer unique capabilities for spatial and temporal control over material properties in biomedical applications, in particular, protein delivery. A variety of illumination sources are now available in clinical settings for light delivery into complex tissues, expanding the role for photoresponsive materials as implantable depots in buried tissue.^[17,18] In notable examples from the Anseth group, photoresponsive hydrogels have been used to modulate cell behaviour^[19] and for therapeutic drug delivery.^[17] Similarly, the Garcia group employed photocaged peptides to modulate cellular adhesion to a hydrogel scaffold in vivo, and showed that delayed presentation of adhesion peptides reduces inflammation and fibrosis in living tissue.^[20]

In general, photoresponsive hydrogels make use of o-nitrophenyl or coumarin-based photocleavable moieties, either as a caging group to block peptide activity, or as a degradable crosslinker to modulate mechanical properties. These organic moieties can be synthetically difficult to incorporate and respond most sensitively to near-UV (350 – 400 nm) light, which has minimal tissue penetration due to scattering and absorption.^[21] Recent advances have demonstrated the delivery of proteins cued by longer wavelengths (600 – 900 nm) using a red-shifted azobenzene guest-β-cyclodextran host interaction or upconverting nanoparticles,^[22,23] and the development of a hydrogel that responds rapidly to near-UV light.^[24] However, there remain significant challenges to combine longer-wavelength-absorbing phototriggers with rapid hydrogel responses, as well as improving chemical syntheses in such materials systems.

To generate an efficient, visible-light-responsive hydrogel system, we synthesized a photodegradable crosslinker, [Ru(bpy)₂(3-pyridinaldehyde)₂]Cl₂ (RuAldehyde, Figure 1a), designed to react in a simple 1-step procedure with hydrazide-modified hyaluronic acid (HA-HYD) to form a Ru-hydrogel. This system takes advantage of the rich photochemistry and ready synthesis of Ru(II) polypyridyl complexes,^[25–28] as well as the well-known biocompatibility of HA, which has been FDA-approved for use in many biomedical applications.^[29,30] Previous systems have successfully used ruthenium compounds, often coupled with upconverting nanoparticles, to achieve near-IR activation of the ruthenium and release of active protein.^[31,32]

Figure 1.

RuAldehyde structure and photochemistry. a) Visible light (<530 nm)-induced ligand exchange with water. The measured distance between aldehyde carbons is 5.6 Å (crystal structure reprted in Figure S1). b) UV-Vis spectra of the photolysis of RuAldehyde (λ_ex = 450 nm, 14 mW/cm²).

Ru(bpy)₂L₂ complexes such as RuAldehyde, where bpy is 2,2′-bipyridine, and L is a pyridine-based ligand, have been shown in the literature to undergo substitution of a single pyridine in water to yield Ru(bpy)₂L(H₂O) and free ligand L,^[33,34] a process which occurs in less than 20 ns (Figure 1a).^[34] RuAldehyde visible absorbance (λ_max = 450 nm, ε₄₅₀ = 6400 ± 300 M^-1cm^-1) is indicative of a MLCT band, commonly seen in ruthenium polypyridyl coordination complexes. RuAldehyde exhibits strong absorbance out to 500 nm with a tail extending past 530 nm (Figure 1b).

Monitoring the photolysis reactionby UV-Vis spectroscopy, we observed red-shifted absorbance consistent with pyridine-to-water ligand exchange.^[35] A single product peak with one isosbestic point (455 nm) was observed, suggesting complete photolysis of only one Ru – pyridine bond.^[36] This model and the ¹H NMR and ESI data (Figures S2, S3) identify the primary photolysis product as Ru(bpy)₂(3-pyridinaldehyde)(H₂O) (Figure 1b). Thus, the Ru photoproduct remains attached to HA post irradiation, which is advantageous for biological applications of Ru-HA hydrogel.

Absorbance changes for RuAldehyde irradiation at 450 nm were fit to a single ligand substitution, pseudo-first-order kinetic model (Figure S4). From this model, we determined the quantum yield of 3-pyridinaldehyde (3-pa) photorelease: Φ_pr = 0.63 ± 0.01. High quantum yield, coupled with large absorptivity in the visible region, give RuAldehyde unique photophysical properties. Efficiency (Φ_pr•ε) of 4.0 × 10³ M^-1cm^-1 at 450 nm is a large improvement over commonly employed nitrobenzyl- or coumarin-based photodegradable linkers with efficiencies of 700-1100 M^-1cm^-1 at near-UV wavelengths.^[17]

To generate hydrogels, RuAldehyde was mixed with HA-HYD at a 1:2 molar ratio of RuAldehyde to hydrazide, to promote complete crosslinking (Scheme 1), with a final concentration of 3 wt% HA-HYD and a corresponding final concentration of 13.6 mM RuAldehyde. This formulation allowed the formation of robust hydrogels from low-viscosity solutions. Rheometric data provided insight into the crosslinking kinetics and photodegradation of the hydrogel (Figure 2a). Gelation occurred rapidly, with full crosslinking occurring over approximately 10 min. Upon irradiation with Hg lamp (400-500 nm bandpass filter, 14 mW/cm²), the storage modulus of the sample decreased, dropping from approximately 4000 Pa to < 200 Pa in less than 60 s, with an even faster response observed at higher light intensities. A continued decrease in storage modulus to less than 20 Pa was observed under continuous irradiation, where the samples were observed to degrade into a viscous liquid with no significant elastic properties.

Scheme 1.

Crosslinking hydrazide-modified HA (HA-HYD) with RuAldehyde, followed by visible-light degradation. Ruthenium photoproducts (red) remain attached to HA polymers (blue) after irradiation.

Figure 2.

Hydrogel photodegradation. Hydrogels of hyaluronic acid crosslinked with RuAldehyde show rapid degradation upon irradiation with visible (400-500 nm) light, as illustrated in a) rheological profiles showing a rapid loss of storage modulus (G′) upon irradiation. b) Complete degradation of a macroscopic 4 × 5 mm hydrogel was observed within 50 min using 28 mW/cm² light intensity. c) Thin, 0.5 mm, hydrogels loaded with Texas Red © dextran (45 kDa, 1 mg/mL) were irradiated for 2 min through a photomask with varying intensities of visible light. Higher intensities penetrated deeper into the hydrogel.

The high molar absorptivity of RuAldehyde limits light penetration and slows degradation of larger Ru-hydrogels. A large 5 mm tall by 4 mm wide hydrogel formed on the benchtop took nearly 50 min of illumination to fully degrade using Hg lamp (400-500 nm bandpass filter, 28 mW/cm² light, Figure 2b), whereas somewhat smaller hydrogels (0.5 mm × 4 mm) required 8 min to degrade (Figure 3).The selective masked irradiation of macroscale (0.5 × 4 mm) hydrogels using various intensities of visible light showed deeper etching with higher intensity light after the same time of light exposure (Figure 2c).A similar but diminished effect was observed when 523 nm light was used in the same experiment, due to the lower quantum yield at longer wavelengths (0.15 ± 0.05 @ 532 nm) and the lower absorptivity (Figure S5). These same hydrogels were opaque to blue light (Figure S6). Therefore, hydrogels used for in vitro tests were kept to a thickness of ∼500 μm, which enabled handling while minimizing the time needed for complete degradation. The light intensity and time of irradiation needed to degrade RuAldehyde-crosslinked hydrogels compares favourably to established photodegradable systems, which vary from 60 mW/cm² for 30 min for an ~5 mm gel using the red-shifted azobenzene derivative,^[22] to 40 mW/cm² for 5 min for a coumarin derivatized gel formed on the rheometer (∼30 μm in thickness).^[37]

Figure 3.

Protein photorelease from hydrogels. a) TEM1 protein was encapsulated within hydrogels that were then irradiated (450 nm, 14 mW/cm²) to release active TEM1. b) Hydrogels were irradiated either continuously (orange), in 4-min intervals every hour (green), or left in the dark (black). % activity was determined by activity assay and compared to TEM1 activity prior to encapsulation.

The exchange of only one 3-pyridinaldehyde ligand minimizes the potential toxicity of the hydrogel Ru photoproducts. In this hydrogel system, the doubly ligand-substituted product Ru(bpy)₂(H₂O)₂ is never formed, and the ruthenium photoproduct remains attached to the HA backbone (Scheme 1). No decrease in viability was observed in cells exposed to non-degraded hydrogels or to acute (1 day) exposures to the hydrogel photoproducts from in vitro assays (Figure S8).

When ruthenium-based photoproducts are released from the polymeric backbone significant cell toxicity can be observed, as RuL₅(H₂O) compounds are known ROS generators under irradiation. This was observed by Sun et al. in their block copolymer system designed to release Ru(biquinoline)(terpyridine)(H₂O) from the polymer and induce cell death.^[38] With the lack of ruthenium release in our current system the potential toxicity is mitigated.

The Ru-HA system was explored for protein photo-delivery using macroscale hydrogel depots (discs: 4 mm diameter, 500 μm thick) encapsulating TEM1 β-lactamase (TEM1) as a model protein. TEM1 is a 29 kDa enzyme responsible for antibiotic resistance in bacteria by catalysing the ring-opening of beta-lactam groups present in many common antibiotics.^[39] TEM1 is similar in size to other small bioactive proteins and peptides including human growth hormone (22 kDa) and light chain fragments of some antibody drug targets (24 kDa).^[6,7,40,41] Importantly, TEM1 activity on nitrocefin can be measured using a colorimetric assay at wavelengths that are not affected by residual Ru-HA material in solution.

o retain active enzyme within the hydrogel until light-mediated release Ru-hydrogels loaded with TEM1 (0.5 mg/mL) were incubated briefly in a reducing buffer containing NaCNBH₃ to create stable crosslinks between lysine residues on TEM1 and RuAldehyde (Figure 3a). In the dark, the hydrogels showed < 1%release of TEM1 for up to 5 days (Figure S8).

The possibility for step-wise release of active enzyme in response to intermittent light was tested as well as more rapid, complete release under continuous irradiation (Figure 3b). These experiments confirmed that light exposure modulates release, enabling consistent step-wise dosing of an active protein from Ru-hydrogel via light-mediated surface erosion. Because one RuAldehyde ligand is exchanged, TEM1 protein released from the hydrogel was modified with residual ruthenium complex as Ru(bpy)₂(H₂O)(3pa-protein) or with 3pa ligand alone (Figure 3a).

It has been demonstrated for several proteins that physical crosslinking to gel matrices can minimally impact biological activity,^[42,43] and may result in somewhat decreased activity for TEM1 released from Ru-hydrogels (Figure S8).

Microfluidic processing of Ru-hydrogels into microgels offered the possibility to combine the versatility of this photoresponsive material system with the strengths of delivery vehicles generated using microfluidics.^[44] These include, for example, greater uniformity in size and mechanical characteristics and ease of injectability and implantability. An additional goal was to produce microgels capable of complete degradation upon very short exposures to light. in the collected microparticle emulsion.

Because of the rapid, but not instantaneous, gelation kinetics resulting from the use of the pyridine-aldehyde group as opposed to an aliphatic aldehyde,^[45] gelation times allowed mixing of the hydrogel components in a microfluidic mixing device (Figure 4a),^[46,47] with gelation and curing occurring

Figure 4.

Generation of microgels with rapid degradation properties. a) Microfluidics were used to combine materials in aqueous droplets that were dispersed in mineral oil and mixed in curved channels to form uniform microgels, which were subsequently washed into an aqueous medium. b) Microgels in PBS were exposed to 10 mW/cm² light for 5 s, resulting in microgel degradation, which is shown with time post-irradiation indicated. c) Complete degradation, determined from Ru-bound HA released, occurred within 60 s irradiation.

Microgels had an average diameter of 74 ± 6 μm (n = 100 particles) as determined by optical microscopy. Microgels suspended in phosphate buffered saline that were exposed to 10 mW/cm² for 5 s on the microscope stage were observed to experience rapid dilation and degradation (Figure 4b). Complete particle degradation and Ru-HA release occurred within 60 s of irradiation at 10 mW/cm² (Figure 4c). The microgel-Ru-crosslinker format thus offers the potential for rapid release of payloads with low doses of visible light.

In summary, a photoactive ruthenium crosslinker enabled the creation of hydrogels that respond with unique speed and efficiency to visible light exposure. Ru-hydrogels were demonstrated as vehicles for the encapsulation and controlled delivery of viable protein with low doses of visible light, and produced in both macro- and microgel formats. The strong potential exists for tuning the polypyridyl coordination sphere on the ruthenium to develop new RuAldehyde crosslinkers that are responsive to additional visible wavelengths.^[48,49] In this way, we envision ruthenium-crosslinked polymer systems that provide even greater spatiotemporal control over materials properties such as storage modulus and porosity, as well as regulation of drug delivery profiles and cellular function in biomedical applications.