Highly porous and injectable hydrogels derived from cartilage acellularized matrix exhibit reduction and NIR light dual-responsive drug release properties for application in antitumor therapy

The study addresses the need for safer and more effective local chemotherapy delivery to tumors by developing minimally invasive, injectable, and stimuli-responsive hydrogels. Conventional chemotherapy causes severe systemic side effects, and traditional hydrogels often require surgical implantation and have dosing limitations. Extracellular matrix (ECM)-based biomaterials offer biocompatibility but can be mechanically weak and rapidly degrading; many chemical cross-linkers used to reinforce ECM exhibit cytotoxicity. Tumors display elevated redox potential (10–100× higher than healthy tissue), enabling redox-triggered drug delivery. Diselenide bonds, with lower bond energies than disulfide, can cleave under reducing conditions and in response to reactive oxygen species (ROS), offering faster responsiveness. Near-infrared (NIR) light can noninvasively penetrate tissue and, with a photosensitizer such as indocyanine green (ICG), generate ROS to trigger release. However, existing diselenide systems often suffer poor water solubility and insufficient bioorthogonality. This work proposes CAM-based hydrogels cross-linked via a water-soluble diselenide-bearing tetrazine cross-linker and Nb–Tz inverse electron-demand Diels–Alder (IEDDA) chemistry to achieve dual responsiveness (reduction and NIR/ROS) for on-demand, localized drug release in antitumor therapy.

Injectable hydrogels have been developed from natural and synthetic polymers through sol–gel transitions, in situ polymerization, and physical/chemical cross-linking, with ECM-based materials offering low inflammation, biocompatibility, biodegradability, and cell-adhesive ligands. Mechanical weakness and fast degradation of ECM hydrogels have been tuned with cross-linkers such as formaldehyde, glutaraldehyde, genipin, and EDC, though several are cytotoxic. Stimuli-responsive hydrogels have been engineered to respond to pH, temperature, redox, light, electrical signals, and enzymes. Tumor microenvironments have elevated redox potential (∼10–100×), enabling selective redox-triggered delivery. Diselenide bonds (Se–Se 172 kJ/mol; C–Se 244 kJ/mol) are more labile than disulfide (S–S 268 kJ/mol), allowing faster reduction responsiveness and also ROS sensitivity. NIR-responsive systems include organic chromophores (o-nitrobenzyl esters, coumarins, ICG) and inorganic photothermal agents (antimony nanopolyhedra, selenium-coated tellurium nanoheterojunctions, silicon, black phosphorus). Combining ICG with diselenide linkages enables NIR-triggered ROS-mediated cleavage. Yet, many diselenide-based cross-linkers are poorly water-soluble and require toxic solvents, catalysts, or additives, limiting bioorthogonal hydrogel formation.

Materials, instruments, and measurements are detailed in Supplementary Information. CAM extraction: Cartilage acellularized matrix (CAM) was prepared via an optimized decellularization protocol removing cells and nucleic acids while preserving collagen and glycosaminoglycans (GAGs). CAM functionalization: CAM was modified with norbornene (Nb) groups through a two-step carbodiimide coupling strategy. First, 5-norbornene-2-carboxylic acid was converted to its NHS active ester (EDC/NHS in dry DCM at 0 °C then RT; 89% yield). CAM powder was dispersed in bicarbonate buffer (pH 9), and the Nb–NHS ester in DMSO was added (50 mol% relative to free amines). After 48 h at RT, the reaction was quenched with hydroxylamine, precipitated with ethanol, dialyzed (MWCO ∼14 kDa), and lyophilized to afford CAM–Nb. Degree of substitution (ninhydrin assay) was 32% of original amines. Diselenide cross-linker synthesis: Water-soluble diselenide-di-PEG (DSe–DPEG) was synthesized by generating Na2Se2 in water (Se reduced by NaBH4 at 0 °C; additional Se, heated to 100 °C under N2) followed by nucleophilic substitution with bifunctional PEG-OTs (a-tosyl-ω-hydroxy PEG). The product was purified by extraction, recrystallization, and flash chromatography (57% yield). 1H NMR confirmed structure. DSe–DPEG–DTz was synthesized by activating tetrazine(benzylamino)-5-oxopentanoic acid (Tz–COOH) with EDC/DMAP in DCM/DMSO at 0 °C, then coupling to DSe–DPEG under inert atmosphere (48 h, RT), workup and column chromatography (59% yield). 1H NMR confirmed structure. Hydrogel formulation: Injectable hydrogels were formed by mixing CAM–Nb (2% or 4% w/v in PBS) with DSe–DPEG–DTz in varying Nb:Tz feed ratios (10:5 or 10:10). Mixtures were vortexed (10 s) and loaded into 25G syringes; gelation time was measured via inverted vial test. Porosity was visualized by incorporating rhodamine-G6 and imaging by confocal laser scanning microscopy; morphology further assessed by FE-SEM. Viscoelasticity was characterized by rheometry (details in SI). Drug loading and release: DOX-loaded and DOX+ICG-loaded hydrogels were prepared by premixing DOX (1 mg/mL for 2% CAM–Nb; 2 mg/mL for 4% CAM–Nb) with or without ICG (1 mg/mL) before cross-linker addition. Loading efficiency was determined by washing hydrogels with PBS and quantifying DOX in supernatants by UV–Vis (λmax 485 nm) against standard curves. In vitro release was tested in PBS (pH 7.4), PBS with 10 mM GSH (reducing), and in PBS for DOX+ICG hydrogels after 15 min NIR irradiation (2 W). Lyophilized hydrogel disks were placed in dialysis bags (3.5 kDa MWCO) in 30 mL medium at 37 °C, 100 rpm, sampling over time with replenishment; cumulative release quantified by UV–Vis. Cytocompatibility: HFF-1 fibroblasts were exposed to CAM and CAM–Nb (100–2000 µg/mL) for 48 h; viability measured by WST assay; live/dead assessed with calcein-AM/ethidium bromide staining. Empty hydrogel cytocompatibility was tested in HT-29 cells by WST. Antitumor activity: For GSH-triggered release, HT-29 cells were pre-incubated with 10 mM GSH for 1 h, then treated with DOX-loaded hydrogels or free DOX for 48 h; viability assessed by WST. For NIR-triggered release, DOX+ICG hydrogels or free DOX were placed in Transwell inserts above HT-29 cells; samples irradiated with 2 W NIR laser for 90 s, then incubated 48 h prior to WST assay.

CAM characterization: Decellularization preserved collagen (∼400 µg/mg CAM) and 75% of GAGs (∼175 µg/mg CAM) relative to native cartilage; DNA was undetectable; SDS-PAGE showed dominant collagen band at ∼150 kDa. CAM–Nb synthesis achieved a 32% degree of substitution of primary amines, confirmed by FTIR and ninhydrin assay. Hydrogel formation: Rapid, syringe-injectable gels formed via Nb–Tz IEDDA with N2 evolution, producing highly porous structures confirmed by confocal microscopy and FE-SEM. Gelation was tunable by polymer content and Nb:Tz ratio. Formulation metrics (mean ± SD): - CAMHG-1 (2% CAM–Nb, 10:5 Nb:Tz): gel time 478 ± 30 s; DOX loading efficiency 73 ± 6%; loading content 3.65 ± 0.07%. - CAMHG-2 (2% CAM–Nb, 10:10): gel time 121 ± 15 s; loading efficiency 81 ± 5%; loading content 4.05 ± 0.04%. - CAMHG-3 (4% CAM–Nb, 10:5): gel time 72 ± 20 s; loading efficiency 89 ± 2%; loading content 4.51 ± 0.06%. - CAMHG-4 (4% CAM–Nb, 10:10): gel time 32 ± 20 s; loading efficiency 93 ± 3%; loading content 4.64 ± 0.05%. Drug release: In PBS (pH 7.4), DOX release was minimal. Under reducing conditions (10 mM GSH), sustained DOX release exceeded 90% by 96 h, indicating diselenide cleavage-enabled release. With NIR exposure (2 W; DOX+ICG hydrogels), ROS generation led to a burst release (>50% in first 4 h) followed by sustained release. Biocompatibility and bioorthogonality: CAM–Nb precursor and empty hydrogels were essentially non-toxic to HFF-1 fibroblasts and HT-29 cells by WST and live/dead assays. Antitumor efficacy: DOX-loaded and DOX+ICG-loaded hydrogels, upon GSH or NIR triggering respectively, significantly reduced HT-29 metabolic activity, achieving antitumor effects comparable to free DOX.

The developed CAM-based hydrogels fulfill the design goal of a minimally invasive, bioorthogonal, and dual-stimuli-responsive drug delivery system. The Nb–Tz IEDDA cross-linking proceeds rapidly in aqueous buffer without toxic catalysts, and N2 evolution generates a highly porous network beneficial for mass transport and loading. Mechanical and gelation properties were tunable via CAM concentration and cross-link density, enabling control over injectability and network formation. The diselenide-containing cross-linker confers strong responsiveness to intracellularly relevant reductive conditions (10 mM GSH) and to NIR-induced ROS in the presence of ICG, providing on-demand drug release while minimizing leakage under physiological conditions. High DOX loading efficiencies (up to 93%) and near-complete release in reducing environments translate into robust in vitro antitumor activity against HT-29 cells, comparable to free DOX, while the hydrogel matrix and precursors show cytocompatibility with both fibroblasts and cancer cells in the absence of triggers. Collectively, these results support the feasibility of CAM-based, dual-responsive injectable hydrogels for localized chemotherapy with improved safety and spatiotemporal control.

This work introduces CAM-derived injectable hydrogels cross-linked via water-soluble, diselenide-bearing tetrazine linkers and Nb–Tz IEDDA chemistry, yielding highly porous, cytocompatible networks with tunable gelation and mechanical properties. The systems exhibit dual responsiveness: minimal drug leakage under physiological conditions, sustained reduction-triggered release (>90% DOX at 96 h in 10 mM GSH), and NIR/ROS-triggered burst-plus-sustained release when co-loaded with ICG. DOX-loaded hydrogels achieve antitumor activity against HT-29 cells comparable to free DOX upon appropriate stimuli. These attributes underscore the potential of the developed hydrogels as promising candidates for minimally invasive, on-demand, localized cancer therapy.