Self‐Healing and pH‐Robust Hydrogels Enabled by Synergistic Supramolecular Interactions for Injectable Gastrointestinal Wound Repair

ABSTRACT

Injectable self‐healing hydrogels are promising biomaterials for minimally invasive internal wound repair. However, most hydrogels fail in the gastrointestinal tract, where extreme pH fluctuations, from strong acidity to mild alkalinity, disrupt conventional cross‐linking interactions and lead to network dissociation. In this work, we report an injectable self‐healing and pH‐robust hydrogel dressing that withstands diverse gastrointestinal conditions by leveraging cooperative supramolecular interactions. Specifically, the integration of π‐cation‐π and hydrophobic interactions imparts the hydrogel with exceptional thermo‐responsiveness, high injectability, rapid in situ gelation property, and robust self‐healing capability, independent of environmental fluctuations. Furthermore, the hydrogel effectively resists bacterial adhesion and prevents biofouling, owing to the strong hydration shell formed by the hydrophilic chains within the network. In vivo studies using rat models demonstrate that the hydrogel readily adapts to both gastric and intestinal wound sites, simplifying the surgical procedure compared with sutures and commercial barrier films, which significantly reduces postoperative complications such as adhesion and inflammation while accelerating the healing of gastrointestinal perforations. This work establishes a supramolecular design strategy for engineering multifunctional, environment‐adaptive biomaterials, opening new avenues for internal wound repair and other challenging biomedical applications.

A pH‐robust injectable hydrogel enabled by synergistic supramolecular interactions is developed to address the intrinsic limitations of conventional hydrogel dressings in complex gastrointestinal environments. By actively leveraging environmental stimuli to regulate functionality, this supramolecular synergy strategy provides a versatile platform for designing adaptive polymer‐based biomaterials for advanced biomedical applications.

1. Introduction

Developing advanced injectable self‐healing hydrogels hold promising prospects for minimally invasive therapies due to their simple administration approach, tunable fit‐to‐shape mechanical properties and the ability to recover the structural integrity after injection or inflicted damage [1, 2, 3, 4]. One representative example is the hydrogel dressings for inner wound repair and tissue regeneration [5, 6, 7, 8]. In situ gelation of the hydrogels can fully cover on the irregular and/or heavily folded injured tissues, achieving hemostasis, providing reliable protective barrier against microbial attachment and reducing the risk of postoperative complications such as delayed bleeding, infection and local postoperative adhesion [9, 10, 11, 12, 13]. Current existing injectable self‐healing hydrogel dressings are generally fabricated based on reversible interactions such as dynamic covalent bonds (e.g., Schiff base, boronic ester and disulfide bonds) or salt bridge (i.e., hydrogen bonding and electrostatic interaction) [14, 15, 16, 17]. However, for gastrointestinal perforation (GP), a severely life‐threatening medical emergency associated with considerable mortality of 30%–50% [18, 19, 20, 21], most of these hydrogels lack universal adaptability to the perforation sites, because the cross‐linking interactions fail to be consistently reversible in varied gastrointestinal environments (e.g., a series of pHs from strong acidicity to weak alkalinity, high salinity) (Figure 1A, B) [22, 23, 24]. For example, the dynamic covalent bonds, though applicable at slightly alkalinity, are subjected to hydrolysis under acidic environments [25, 26, 27]. Hydrogen bond often presents a difficulty for maintaining stability at various pHs as the receptor deprotonation or protonation would engender bonding dissociation [4, 28]. Electrostatic interaction is highly suppressed owing to the dramatically shortened interaction range (assessed by the Debye length) under physiological salinity conditions [29, 30]. Thus, the local environment‐triggered broken and/or weakened bonding interactions would inevitably induce the irreversible decomposition of the hydrogel network, causing the wound sites exposed to surrounding environments, which thereby result in postoperative complications to retard or even impede wound healing [7, 31].

FIGURE 1.

(A) pH values of different organs in gastrointestinal tract. (B) Weakness of the conventional injectable hydrogel dressing for gastrointestinal perforation treatment in complex gastrointestinal environments. (C) Ideal injectable self‐healing hydrogel wound dressing with universal adaptability to complex gastrointestinal environment for gastrointestinal perforation treatment. (D) Chemical structure of an ABA triblock copolymer that designed for preparing the universal hydrogel wound dressing with the hydrogel 3D network being constructed via copolymer assembly driven by the synergy of π‐cation‐π and hydrophobic interactions.

To guarantee the universal adaptability of injectable self‐healing hydrogel dressings for GP treatment, the reversibility of the interactions driving the hydrogel network formation needs to be insusceptible to complex gastrointestinal environments [4, 32, 33]. To be specific, salt‐ and pH‐independent interactions may hold the key to ensuring hydrogel cross‐linking since high salinity and wide pH range are the most typical features of the complex biological environments in gastrointestinal tract [7, 28, 34]. Hydrophobic interaction and π‐interactions are two representative reversible non‐covalent interactions capable of exhibiting salt‐ and pH‐independent (or only slightly dependent) behaviors in certain material systems with specific molecule design [35, 36]. Hydrophobic interaction‐enabled injectable hydrogels have been fabricated by copolymerizing hydrophobic monomers that assembled in ionic surfactant‐based micelles with hydrophilic monomers, in which the hydrophobic association between the alkyl pendants of copolymers and the alkyl tails of surfactants acted as reversible cross‐links responsible for self‐healing [37, 38]. Cation‐π interaction, as the common type of π‐interactions, has presented insensitivity to salinity and pH in material structures designed with quaternary ammoniums and aromatic groups, the strong and reversible properties of which confer the as‐prepared hydrogels with robust self‐recovering ability [39, 40]. Nevertheless, the ionic surfactants or quaternary ammoniums, serving as the functional ingredients for the self‐healing processes, usually associate with cytotoxicity issues, rendering hydrogels with inferior biocompatibility and restricting their biomedical applications [41, 42]. Reducing the ingredient concentrations may be beneficial in alleviating cytotoxicity, but at the expense of sacrificing the cross‐linking interaction strength [43, 44]. Given that such environment‐independent self‐healing behavior mainly rely on passively preventing the reversible interactions in hydrogel network from being weakened by the surroundings, the impaired interaction strength will inevitably compromise the self‐healing effectiveness. To address these limitations, biocompatible hydrogel materials that can actively and skillfully leverage diverse local environments to improve their in situ‐gelling and self‐healing properties would be ideal as the wound dressings for complex gastrointestinal tract.

Herein, we report a paradigm of injectable self‐healing hydrogel with universal adaptability to diverse gastrointestinal circumstances based on the complex environment‐promoted self‐assembly of an ABA triblock copolymer, and have substantiated its applicability as wound dressing for GP healing (Figure 1C). The ABA triblock copolymer was designed and synthesized by copolymerizing salt cation‐responsive π donator component of benzyl methacrylate (BM) and thermal‐responsive N‐isopropylacrylamide (NIPAM) as terminal blocks onto both flanks of the middle hydrophilic poly(ethylene glycol) (PEG) block, denoted as P(BN‐PEG‐NB) (Figure 1D). Hydrophobic interaction between the NIPAM components that dehydrated at body temperature, acting as physical cross‐linking linkages, rendered the copolymer solution with rapid thermo‐responsive sol‐to‐gel transition when injected into physiological surroundings [45, 46]. Meanwhile, by smartly utilizing the free salt cations (i.e., Na⁺), a π‐cation‐π interaction arisen from the BM components was orchestrated into the NIPAM‐generated hydrophobic domains [47]. Through the nanoscale intermolecular force measurements and macroscale rheological tests, the π‐cation‐π interaction was identified to be pH‐independent, and to cooperate with the hydrophobic interaction to significantly enhance the hydrogel self‐healing capability with insusceptibility to complex gastrointestinal tract. Owing to a large amount of strongly hydrated PEG blocks functioning as bridges in the 3D copolymer network [48], such P(BN‐PEG‐NB)‐assembled hydrogel exhibited robust resistance against biofoulants under diverse physiological conditions. In a rat model, the hydrogel could be easily delivered to the perforation sites of the stomach and intestine through simple injection and could readily adapt to these two distinct local environments. Compared with conventional GP treatment, the in situ‐assembled hydrogel dressing offered advantages on simplifying the surgical process, preventing postoperative tissue adhesion, reducing inflammation and promoting wound healing in complex gastrointestinal system. The realization of synergizing multiple supramolecular interactions to tune hydrogel properties as a universal wound dressing for GP repair paves a promising route for designing hydrogel‐based biomaterials with customized functions from molecular scale interaction mediation.

2. Results and Discussion

The ABA triblock copolymer poly[(N‐isopropylacrylamide)‐co‐(benzyl methacrylate)]‐b‐poly(ethylene glycol)‐b‐poly[(N‐isopropylacrylamide)‐co‐(benzyl methacrylate)], P(BN‐PEG‐NB), was synthesized through the reversible addition‐fragmentation chain transfer (RAFT) polymerization (Figure S1A). The synthesized copolymer was characterized by proton nuclear magnetic resonance (¹H NMR) and gel permeation chromatography (GPC) (Figure S1B and Table S1). The hydrogels were separately prepared by dissolving P(BN‐PEG‐NB) copolymer in 150 mm NaCl with a series of pHs (i.e., pH 2.5, 5.4, 7.4, 8.5) or 150 mm PBS buffer at a copolymer concentration of 5 w/v%. The as‐prepared P(BN‐PEG‐NB) hydrogels were named as NaCl‐rich P(BN‐PEG‐NB)@pHs (including ‐@pH2.5, ‐@pH5.4, ‐@pH7.4, ‐@pH8.5) and NaCl‐rich P(BN‐PEG‐NB)@PBS hydrogels, respectively. The optical images show the fully reversible sol‐gel transition of P(BN‐PEG‐NB) samples between 4°C and 37°C under all pH conditions (Figure 2A and Figure S2), implying that the hydrogels were constructed via physical interactions rather than heat‐induced non‐reversible chemical reactions [49]. Their thermo‐sensitivities were quantitatively investigated through the temperature ramp rheological measurements, in which the storage modulus G′ and loss modulus G′′ were recorded with the temperature gradually increasing from 4 to 40°C and maintaining at 40°C for 120 s (Figure 2B). For all pH cases, G′′ was constantly larger than G′ at lower temperature, signifying the hydrogels with liquid‐like behavior. During the heating process, G′ drastically increased and began to exceed G′′ at ∼7°C, the temperature of which was identified as the sol‐gel transition temperature. All G′ reached equilibrium values that were much higher than G′′ with temperature over ∼20°C and stably maintained at the plateau over time at 37°C, suggesting that the gelation occurring in copolymer networks could enable the solid‐like structure integrity of the hydrogels at body temperature. Given the remarkable thermo‐sensitivity and pH insensitivity of the system, injecting the copolymer solutions stored at 4°C into 37°C aqueous media containing 150 mm NaCl at different pH values immediately triggered gelation and the formation of stable hydrogels (Figure 2 Ci and Figure S3). Moreover, any designated shapes such as letters of “SEU” could be facilely “written” by injecting the copolymer solution onto a 37°C platform mimicking tissue temperature (Figure 2Cii). Such injectability endows the designed hydrogel dressing with potential ability of delivering to wound sites with intricate shapes through laparoscopic surgery.

FIGURE 2.

Evaluation of the thermo‐sensitive and self‐healing properties of P(BN‐PEG‐NB) hydrogels prepared under various pHs. (A) Optical photographs of the reversible sol‐gel transition of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels triggered by temperature at pH 2.5. (B) Thermo‐responsive storage (G′) and loss (G′′) modulus variation of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels. (C) i. Injecting 4°C‐stored liquid state NaCl‐rich P(BN‐PEG‐NB)@pH2.5 hydrogel sample into 37°C aqueous solution (pH 2.5, 150 mm NaCl) from a 23‐gauge needle, and ii. the letters “SEU” written on a 37°C platform through injection. (D) Optical photographs of the self‐healing processes of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels at 37°C. (E) Oscillatory strain sweep measurements of G′ and G′′ of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels at 37°C as a function of strain γ (left) followed by time sweep measurements with the immediate recovery of γ from 1000% to 5% (right). (F) Dynamic alternate strain step measurements (γ = 5% or 650%) of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels at 37°C to evaluate the self‐healing performance. (G) Viscosity measurements of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels at 37°C as a function of shear rate. (H) Comparison of self‐healing efficiency (G′ modulus recovery) between NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels and salt‐free P(BN‐PEG‐NB) hydrogels (without adding 150 mm NaCl) at 37°C. (I) Oscillatory strain sweep measurements of G′ and G′′ of LiCl‐rich P(BN‐PEG‐NB)@pHs hydrogels at 37°C as a function of strain γ (left) followed by time sweep measurements with the immediate recovery of γ from 1000% to 5% (right).

To serve as the qualified wound dressing for GP, it is essential for the hydrogels to be adapt to complex gastrointestinal environments and to rapidly self‐heal from repeated damages to guarantee their functionalities. As shown in Figure 2D, for each of the NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels, two cutting‐off hydrogel fragments could instantly adhere together and autonomously heal into one integral piece when brought into contact at body temperature (i.e., 37°C). Their self‐healing capabilities were evaluated through strain sweep rheological measurements. Figure 2E shows that G′ and G′′ were initially constant within the strain amplitude γ ∼60%, but dramatically dropped with further increasing the strain. The intersection of G′ and G′′ occurred at γ ∼600%, indicating the disruption of the hydrogel networks beyond the critical strain point. It is worth noting that these hydrogels could completely restore and retain their structure integrity when γ immediately turned back to 5% after severe strain deformation of 1000%. Dynamic alternate strain step tests (γ = 5% or 650%) were also applied on these NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels (Figure 2F). All G′ decreased from ∼1200 Pa at γ = 5% to ∼20 Pa at γ = 650%. However, once γ returned to 5%, both G′ and G′′ fully restored the initial values. The recovery process is almost instant and fully reversible during multiple cycles of alternation, implying that polymer chains in ruptured hydrogel structures should be physically dislocated and could rapidly self‐assemble to re‐form the network bridges. The viscosity of these hydrogels at 37°C and 5% strain was observed to be sharply reduced with increasing the shear rate (Figure 2G). This shear‐thinning behavior could render the polymer chains with high mobility after dislocation to boost their diffusion and self‐assembly, which is beneficial to the self‐healing of these hydrogels [50]. To assess the contribution of free salt ions to such self‐healing performance, 5 w/v% P(BN‐PEG‐NB) hydrogels were prepared in aqueous media at the pHs of 2.5, 5.4, 7.4 and 8.5 (adjustment with diluted acetic acid or diluted sodium hydroxide solution) without adding 150 mm NaCl, denoting as salt‐free P(BN‐PEG‐NB)@pHs hydrogels. Despite displaying the similar thermo‐responsive behavior as NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels (Figure S4), the storage modulus (G′) and self‐healing efficiency of the salt‐free P(BN‐PEG‐NB)@pHs hydrogels are different from that of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels. At body temperature, the G′ values of these salt‐free P(BN‐PEG‐NB)@pHs hydrogels were ∼800 Pa (Figure S4), which is reduced by about one third compared with that containing 150 mm NaCl (∼1200 Pa). This pronounced decrease in G′ indicates significantly weakened mechanical properties in the absence of ionic crosslinking, highlighting the important role of Na⁺ ions in mechanical reinforcement of the hydrogel network. In addition, in sharp contrast with the almost full recovery of the modulus of the NaCl‐rich hydrogels, the modulus recovery of the salt‐free hydrogels could only reach ∼62% of their initial values after being damaged (Figure 2H). revealing the importance of salt ions during the self‐healing process. To prove that the salt‐induced remarkable self‐healing performance was mainly due to the cations (i.e., Na⁺) rather than the anions (i.e., Cl⁻), 150 mm LiCl was employed to prepare Li‐rich P(BN‐PEG‐NB) hydrogels at various pHs (viz., Li‐rich P(BN‐PEG‐NB)@pHs hydrogels). As shown in Figure 2I, the G′ values of Li‐rich P(BN‐PEG‐NB)@pHs hydrogels were ∼850 Pa and their modulus recovery efficiency was ∼65% at 37°C, comparable with that of salt‐free P(BN‐PEG‐NB)@pHs hydrogels. It has been reported that Li⁺ possesses very limited binding affinity to π‐conjugated BM moieties of the ABA copolymer than that of Na⁺ [39, 51, 52]. Therefore, the decreased mechanical strength and self‐healing capacity of the LiCl‐rich and salt‐free P(BN‐PEG‐NB)@pHs hydrogels are most likely ascribed to the weakening or loss of π‐cation‐π bridging within the copolymer network, highlighting the synergistic interplay between thermo‐enhanced hydrophobic interactions and cation‐induced π‐cation‐π interaction in reinforcing the self‐healing ability of P(BN‐PEG‐NB) hydrogels.

As it is well‐known that the self‐healing behavior of hydrogel is fundamentally governed by the intermolecular interactions within its polymer network [49, 53]. Strong and reversible supramolecular interactions between the functional moieties that serve as cross‐linking sites on the polymer chains can greatly promote the self‐healing of hydrogels from repeated damage [54, 55, 56]. Thus, to elucidate the outstanding self‐healing mechanism of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels, it is essential to quantify the intermolecular interactions within the ABA triblock P(BN‐PEG‐NB) copolymer networks in gastrointestinal‐mimicking aqueous environments. Considering that NIPAM and BM moieties were the components of A blocks responsible for the physical cross‐linking and self‐healing of P(BN‐PEG‐NB) hydrogel, these two monomers were utilized to synthesize a random copolymer named as P(NIPAM‐co‐BM) (Figure 3A), in which the monomer ratio was kept almost consistent with that in P(BN‐PEG‐NB) (Figure S6 and Table S2). A surface forces apparatus (SFA) was employed to probe the interaction forces between P(NIPAM‐co‐BM) copolymers under various aqueous conditions (i.e., aqueous solutions at different typical gastrointestinal pHs with or without adding 150 mm NaCl) at room temperature (i.e., 20°C). The normalized force‐distance profiles (F/R vs D) and cohesion (F _co/R) were obtained during the approach and separation of two P(NIPAM‐co‐BM)‐coated surfaces. The SFA experiment setup and the atomic force microscopy (AFM) morphology of the smooth and compact P(NIPAM‐co‐BM) coating are depicted in Figure 3B left and right, respectively. As shown in Figure 3C–F, for the testing solutions at all pHs without adding 150 mm NaCl, no obvious strong long‐range repulsions were detected during surface approach, revealing that the NIPAM component of the coating would not form the hydrophobic aggregates to generate steric repulsion at room temperature. When the surfaces were separated, cohesion of ≈1.1, ≈1, ≈1.4 and ≈1 mN m⁻¹ were measured at pH 2.5, 5.4, 7.4 and 8.5, respectively, possibly pertaining to the weakly attractive hydrophobic and/or π‐π interactions between the benzene rings of BM moieties [57, 58]. In contrast, with introducing 150 mm NaCl, the cohesion at pH 2.5, 5.4, 7.4 and 8.5 notably increased to ≈4.2, ≈4.2, ≈4.4 and ≈4 mN m⁻¹, fourfold stronger than that lack of NaCl. Since all coating thickness D _T/2 were nearly unchangeable before and after the add of NaCl, copolymer structure variation‐induced mechanical interlocks should not account for the cohesion reinforcement. Considering free cations and π‐conjugated BM moieties abounding in the system, the major contribution to such enhanced cohesion is most likely attributed to Na⁺‐participated formation of π‐cation‐π complexes that bridge the π‐conjugated benzene rings from two P(NIPAM‐co‐BM) coatings [47, 59]. The existence of π‐cation‐π interactions was proved by the cation species‐modulated cohesion behavior in Figure 3G, in which the cohesion enhancement follows the trend of cation binding affinity to π moieties [39, 60]. It is worth mentioning that the cohesion increments were reversible as confirmed in three consecutive force measurements and slightly fluctuated around 3 mN m⁻¹ at various pHs (Figure 3H), substantiating very limited influence of pH on π‐cation‐π interaction strength. This pH‐independent robust interaction behavior is critical in preserving the hydrogel with reliable self‐healing performance when applied as the GP wound dressing in different gastrointestinal environments.

FIGURE 3.

Investigation on the nanomechanics underlying the self‐healing behavior of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels. (A) Structure of P(NIPAM‐co‐BM) copolymer for surface decoration. (B) Schematics of intermolecular force measurement using a surface forces apparatus (SFA) and the atomic force microscopy (AFM) topological image of the as‐prepared P(NIPAM‐co‐BM) coating for SFA tests. Normalized force‐distance profiles and cohesion measured between two symmetric P(NIPAM‐co‐BM) coating in 20°C aqueous solutions with/without 150 mm NaCl at (C) pH 2.5 (D) pH 5.4 (E) pH 7.4 (F) pH 8.5. (G) The impact of cation species (salt concentration of 150 mm) on the normalized interfacial cohesion between P(NIPAM‐co‐BM) coatings in 20°C aqueous solutions at various pHs. (H) Assessment of the cohesion reversibility and stability between P(NIPAM‐co‐BM) coatings in 20°C aqueous solutions with 150 mm NaCl at various pHs. (I) Optimized geometry of Na⁺‐methylbenzene pair and methylbenzene‐Na⁺‐methylbenzene complex in vacuum phase. The white, grey, purple balls stand for hydrogen, carbon and sodium atoms, respective. (J) Binding energies of Na⁺‐methylbenzene pair and methylbenzene‐Na⁺‐methylbenzene complex in vacuum and water phase obtained from density functional theory (DFT) simulations. (K) Schematic illustration of local hydrophobic microenvironment‐enhanced π‐cation‐π interaction in P(BN‐PEG‐NB) hydrogel network.

Density functional theory (DFT) simulation was further performed to understand the synergistic effect of local hydrophobic environment and π‐cation‐π interaction on promoting the self‐healing performance of the hydrogel. Benzene ring with one substituted methyl group (methylbenzene) was modeled as the π‐conjugated benzene ring on BM moiety, and the Na⁺ that widely exists in biological surroundings was used as the cation. The DFT simulation was performed in vacuum and water phases to evaluate the impact of surrounding phases on the binding affinities of Na⁺ to methylbenzene. The optimized geometries of binary Na⁺‐methylbenzene and ternary methylbenzene‐Na⁺‐methylbenzene complexes in vacuum phase were, respectively similar to that in water phase (Figure 3I and Figure S7), and the geometries of which are consistent with exemplary cation‐π and π‐cation‐π geometries reported in previous studies. The binding energies of one Na⁺ intercalating into two methylbenzene molecules to form the ternary complex were much stronger than that of the binary pair formed by one Na⁺ and one methylbenzene molecule in both vacuum and water cases (Figure 3J). Thus, the transformation of cation‐π pair to π‐cation‐π complex is energetically favorable, rationalizing the formation of π‐cation‐π bridges in P(BN‐PEG‐NB) network coexisting with Na⁺ for facilitating hydrogel self‐healing property [60, 61]. Notably, the binding strength of the ternary methylbenzene‐Na⁺‐methylbenzene complex formed in vacuum phase (≈ ‐173 kJ mol⁻¹) was over threefold stronger than that in water phase (≈ ‐51 kJ mol⁻¹). Such surrounding phase‐dependent π‐cation‐π interaction strength is closely related to the degree of cation hydration, which can be well interpreted by the desolvation penalty of cations [62, 63]. The well hydrated cations in water phase are compelled to pay large desolvation energy costs to dehydrate before binding with two hydrophobic π‐conjugated methylbenzene molecules. On the contrary, cations in vacuum phase are unhydrated and can directly form π‐cation‐π complex without consuming any desolvation energies. For the hydrogel prepared by P(BN‐PEG‐NB) triblock copolymers, the association of thermo‐responsive NIPAM moieties in the terminal blocks (formed by the copolymerization of NIPAM and BM monomers) at body temperature could establish local hydrophobic microenvironments as the cross‐links in hydrogel network [4, 32, 64]. In aqueous environments, these local hydrophobic domains, though not being able to thoroughly prevent Na⁺ ions from hydrating, could assist these cations to push out the surrounding water molecules and lower their desolvation penalty for π‐cation‐π bridging when the hydrated Na⁺ ions travel into the cross‐links to interact with BM moieties (Figure 3K) [65, 66]. Therefore, the π‐cation‐π interaction formed in local cross‐linking hydrophobic microenvironments should be notably strengthened compared with that in aqueous media, which would be more effective to promote the mechanical strength and self‐healing capacity of the P(BN‐PEG‐NB) hydrogel.

When delivered to the GP sites, the wound dressing is inevitably subjected to external mechanical force due to the gastrointestinal motility such as tonic contraction, receptive relaxation and peristalsis [7]. Thus, the adhesion of the NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels to injured tissues is of great importance to prevent their detachment from the wound sites during the wound healing process. To evaluate their adhesion performance, 4°C‐preserved P(BN‐PEG‐NB) solutions were spread on a series of wet porcine tissues, including stomach, large intestine, skin and small intestine, in vitro at 37°C to examine whether the in situ self‐gelling NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels could maintain intimate attachment with these tissue surfaces after the hydrogel‐coated tissues had undergone various deformations or had immersed in aqueous media for 30 min (Figure 4A). The adhesion stresses to these wet tissues were measured by lap shear tests through injecting the sol state NaCl‐rich P(BN‐PEG‐NB)@pHs samples into between two porcine tissues at 37°C and allowing the in situ self‐gelation of 30 s (Figure 4B). As shown in Figure 4C–F, four in situ formed NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels could tightly adhere to various porcine tissues wetted by 150 mm NaCl aqueous solution with corresponding pH, which could be evidenced by the ability to withstand the deformations including stretching, twisting and bending. Besides, to mimic the working conditions of diverse environments of gastrointestinal tract, the hydrogel‐bearing tissues was totally soaked in 150 mm PBS buffer with varying pH. It could be observed that the adherent hydrogels remained intact on the tissues without any detachment after the immersion of 30 min. In addition, the NaCl‐rich P(BN‐PEG‐NB)@PBS hydrogels with the rectangle shape attached on four wet porcine tissues were immersed in these 37°C media at pH 2.5, pH 5.4, pH 7.4 and pH 8.5, respectively, to investigate their swelling behavior. The swelling rate over time were assessed using a ruler to measure the length of these rectangles (Figure S8). These hydrogels were found to display a pH‐independent swelling behavior and slowly swelled with almost the same speed over time, yet still attached on the porcine tissues and maintained structure integrity, indicating the potential feasibility of the NaCl‐rich P(BN‐PEG‐NB)@PBS hydrogel to form a reliable protective layer on wound site in GI tract. The adhesive strength of these four NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels to various porcine tissues was measured as ∼7.6 kPa for porcine stomach at pH 2.5, ∼7.7 kPa for porcine large intestine at pH 5.5, ∼7.8 kPa for porcine skin at pH 7.4 and ∼7.5 kPa for porcine small intestine at pH 8.5, respectively (Figure 4G), which are relatively stronger than that of the salt‐free P(BN‐PEG‐NB)@pHs hydrogels (Figure S9). Such adhesion was further determined to be reversible through three sequential lap shear tests using the recollected NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels from the tested porcine tissues (Figure 4H), which should be attributed to noncovalent interactions between the hydrogels and porcine tissues [67]. It is well‐known that the hydrophobic amino acids (e.g., phenylalanine, isoleucine and valine) abound in the peptides of porcine tissues [68, 69]. Therefore, the adhesion most likely arises from the pH‐independent hydrophobic interaction of the phenyl and thermo‐responsive isopropyl groups in P(BN‐PEG‐NB) copolymers to the pendant hydrophobic groups in amino acid residues on the tissue (Figure 4I) [70], and the Na⁺‐induced π‐cation‐π interactions between the π‐conjugated BM moieties on P(BN‐PEG‐NB) copolymers and amino acid residues containing aromatic group (e.g., phenylalanine) in biological tissues [33]. The pH‐independent hydrophobic interaction‐dominated adhesion behavior endows the hydrogels great capacity to firmly and stably attach to various wound sites regardless of the varied pH of gastrointestinal environments.

FIGURE 4.

Adhesion performance of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels to various animal tissues in vitro at different pH conditions. (A) Illustration of assessing the attachment ability of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels on various wet tissues after stretching, bending, twisting and total immersion in water media. (B) Schematic diagram illustrating the measurement of adhesive strength of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels to various wet tissues through lap shear test. Intimate attachment of NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels on (C) porcine stomach at pH 2.5 (D) porcine large intestine at pH 5.4 (E) porcine skin at pH 7.4 (F) porcine small intestine at pH 8.5 after undergoing various deformation and soaking in water solutions with corresponding pH. (G) Representative lap shear adhesion curves of various biological tissues adhered by NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels. (H) Adhesion reversibility assessment through three sequential lap shear tests using NaCl‐rich P(BN‐PEG‐NB)@pHs hydrogels recollected from the tested tissue samples. (I) Schematic illustration of adhesion mechanism.

Preventing the adsorption of biofoulants such as biofluids and microbe on the surface of implanted hydrogel wound dressing is essential to avoid undesirable immune response and/or inflammation that would delay the healing process. To evaluate the antifouling performance of the hydrogels, silica substrates coated with P(BN‐PEG‐NB) hydrogel layer were immersed into solutions of representative biofoulants including fetal bovine serum (FBS) and high concentrated bacterial (i.e., E. coil) suspension for 4 h at 37°C followed by being rinsed with DI water. Bare silica substrate surface was utilized as a comparison. As shown in Figure 5A, optical photothermal infrared (O‐PTIR) spectroscopy was employed to characterize the FBS residues remained on the substrates after DI water washing, which is a noncontact technique couples visible and infrared lasers to precisely identify biofoulants according to their characteristic IR peaks. The IR scanning images of bare and P(BN‐PEG‐NB) hydrogel layer‐coated silica substrates at the amide peak of FBS (wavelength of 1650 cm⁻¹) displays that, in contrast to a large number of FBS residues almost fully covered on the bare silica surface, only trace FBS moieties sporadically adsorbed on P(BN‐PEG‐NB) copolymer‐coated silica surface. For E. coil, the remanent E. coil bacteria on the as‐tested surfaces were dyed with 4′,6‐diamidino‐2‐phenylindol (DAPI). The fluorescence (FL) microscopy imaging and the relative FL intensity calculation in Figure 5B show only minor signal of E. coil on P(BN‐PEG‐NB) hydrogel layer‐coated silica surface while an obvious E. coil signal on bare silica surface. These antifouling experimental results demonstrate the remarkable reduced biofouling on P(BN‐PEG‐NB) copolymer‐coated surface, which could be attributed to the long‐chain hydrophilic PEG blocks in P(BN‐PEG‐NB) copolymer structure, forming strong hydration defensive layer to inhibit biofoulant attachment [71, 72]. The biocompatibility of the hydrogel dressing was also investigated by assessing the cytotoxicity of P(BN‐PEG‐NB) copolymer using human umbilical vein endothelial cell (HUVEC) and intestinal epithelial cell (IEC) as the model cells, which is another vital property in wound healing. 2D cell culture medium containing P(BN‐PEG‐NB) copolymer was utilized for incubating HUVEC and IEC cells, and the blank cell culture medium was used as the control group. After the incubation of 24 and 72 h, the live and dead cells were stained by calcein and propidium iodide (PI), respectively, to visualize the morphology of cell growth through FL microscopy. As shown in Figure 5C,D, the green FL signal intensity (tained live cells) enhanced with the incubation time increasing from 24 to 72 h, comparable with that of the control group, suggesting the almost undeterred cell proliferation in the presence of P(BN‐PEG‐NB) copolymer. Meanwhile, the copolymer exhibits no significant cell toxicity as evidenced by the unobvious red FL signal (dead cells stained by PI) observed in both cases (Figure S10). The successful evidence of the biocompatibility allows the safe use of P(BN‐PEG‐NB) hydrogel as implantable dressing materials for tissue engineering and regenerative medicine [73].

FIGURE 5.

Anti‐biofouling and biocompatibility tests of P(BN‐PEG‐NB) copolymer. (A) Schematic of optical photothermal infrared (O‐PTIR) working principle, and O‐PTIR imaging on the characteristic peak (amide group of plasma protein 1650 cm⁻¹) of fetal bovine serum adsorbed on bare and P(BN‐PEG‐NB) copolymer‐coated silica substrates. (B) Fluorescence (FL) microscopy images showing the cell nuclei of E. coil adhered on bare and P(BN‐PEG‐NB) copolymer‐coated silica substrates. (C) FL microscopy images of stained live cells of human umbilical vein endothelial cell (HUVEC) and intestinal epithelial cell (IEC) after 24 and 72 h incubation in blank cell culture medium (control group) and P(BN‐PEG‐NB) copolymer‐contained cell culture medium (experimental group). (D) Relative FL intensity calculated from the FL images of stained HUVEC and IEC live cells. Statistical significance was analysis using Minitab Statistical Software (version 21, U.S.) with a Paired t‐test (^* p < 0.05, and ^*** p < 0.001). Data are presented as means ± SD for n = 3 replicates per condition.

Both gastric and intestinal perforations in in vivo rat models were applied to demonstrate the universal capability of the developed P(BN‐PEG‐NB) hydrogel as the wound dressing to treat gastrointestinal perforation in complex biological environments. As shown in Figure 6A,B, three types of repair approaches were employed for the internal wound treatment. The first two were conventional surgical methods including direct suture (ctrl group) and suture‐assisted anti‐adhesive membrane implantation (mem group), and the third one was hydrogel dressing method (P(BN‐PEG‐NB) group) that delivered P(BN‐PEG‐NB) hydrogel via simple injection to form a in situ‐gelling protective layer on wound site. It was observed that the NaCl‐rich P(BN‐PEG‐NB)@PBS solution immediately transformed into stable hydrogel upon injection onto the wound sites of rats, rather than spreading over the surrounding tissue (Figure S11). After 14 days of treatment, the gastrointestinal tissues and the corresponding serum were harvested for further scrutinize including immunohistochemistry (IHC) and enzyme‐linked immunosorbent assay (ELISA). As shown in Figure 6C,D, a macroscopic view on the harvested gastric and intestinal tissues revealed a visible recovery of the wound area treated in P(BN‐PEG‐NB) group, implying the potential feasibility of simple hydrogel dressing administration as an alternative to conventional laboring suturing procedures of ctrl and mem groups for wound healing [74, 75]. Hematoxylin and eosin (H&E) staining showed the distinct difference of the regenerated tissues at the wound sites in three groups in both rat gastric and intestinal perforation models (Figure 6E,F). For the perforations treated with the suture and the membrane implantation, clear gaps or even interruptions were observed in the newly generated seromuscular layers, revealing that the wound sites were only partially healed. On the contrary, the perforations sealed in P(BN‐PEG‐NB) group were completely bridged with the regenerated mucosa, indicating an almost full recovery of the injured tissue. The superior GP repair capacity of the P(BN‐PEG‐NB) hydrogel could be attributed to its ability to maintain the structure integrity via robust reversible cross‐linking interactions in complex gastrointestinal environments, forming a reliable protective hydrogel coating fully covered on the wound site to facilitate wound healing [76, 77].

FIGURE 6.

The internal wound treatment using NaCl‐rich P(BN‐PEG‐NB)@PBS hydrogel dressing in in vivo rat gastric and intestinal perforation models. Macroscopic photographs of (A) gastric perforation and (B) intestinal perforation treated by direct suture (ctrl group), suture‐assisted anti‐adhesive membrane implantation (mem group) and NaCl‐rich P(BN‐PEG‐NB)@PBS hydrogel (P(BN‐PEG‐NB) group). Macroscopic photographs of the (C) gastric and (D) intestinal wounds after 14 days of treatment. H&E staining images of (E) gastric and (F) intestinal wounds harvested on day 14 after three treatment groups. Representative immunofluorescence staining images against cell proliferating marker PCNA (red) at (G) gastric and (H) intestinal wound areas and against angiogenesis marker CD31 (green) at (I) gastric and (J) intestinal wound areas on day 14 after three treatment groups. Quantification on the concentration of pro‐inflammatory cytokines including IL‐1β, IL‐6, and TNF‐α expressed in newly formed capillaries in (K) gastric and (L) intestinal wounds on day 14 after three treatment groups via enzyme‐linked immunosorbent assay (ELISA). Statistical significance was analyzed using Minitab Statistical Software (version 21, U.S.) with one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001). Data are presented as means ± SEM with n = 4 replicates per condition.

The reepithelization at the area of the wound sites was assessed via immunofluorescence staining against 4′,6‐diamidino‐2‐phenylindol (DAPI) and proliferating cell nuclear antigen (PCNA, a marker for cell proliferation) staining (Figure 6G,H). The wound margins and the area of granulation tissues of both gastric and intestinal perforations were visualized by dyeing the cell nuclei with DAPI (blue color), and the proliferating cells were marked by PCNA staining (red color). The merge color of DAPI and PCNA staining (purple) represented the expression of the proteins relating to cell proliferation around the wound sites (i.e., PCNA‐positive cells). The density of PCNA‐positive cells in P(BN‐PEG‐NB) group was observed to be significantly higher than that of ctrl and mem groups (Figure S12), suggesting that P(BN‐PEG‐NB) hydrogel dressing could facilitate cell proliferation during the wound healing [78]. Given that the vasculature regeneration is essential to promote the wound healing process, the neovascularization after the treatment in three groups was evaluated by determining the vascular density at the area of granulation tissue around the wound sites via double immunohistochemical staining against CD31 to mark endothelial cells and angiogenesis and the cell nuclei to locate the wound area [8]. As shown in Figure 6I,J, in both gastric and intestinal perforation models, higher density of positive staining marker for angiogenesis (green color) in P(BN‐PEG‐NB) group could be detected compared with that in ctrl and mem groups. The high vascular density denoted the ability of P(BN‐PEG‐NB) hydrogel dressing for accelerating the formation of vascular networks (Figure S13). The inflammatory response during wound healing process in three treatment groups was also assessed by quantifying the concentration of pro‐inflammatory cytokines including IL‐1β, IL‐6, and TNF‐α expressed in newly formed capillaries via ELISA (Figure 6K,L). The expression of these pro‐inflammatory cytokines was clearly reduced in P(BN‐PEG‐NB) than that in other two groups, indicating that the P(BN‐PEG‐NB) hydrogel could effectively constrain inflammation, which is most likely due to the antifouling capability of the strong hydrated PEG chains in hydrogel network, preventing the accumulation of cell debris, bacteria and other microorganisms that may cause infection [79, 80, 81]. Regarding the clearance pathway of the P(BN‐PEG‐NB) hydrogel after wound healing, as the GI tract is a luminal/open tract with active clearance capacity, the clearance pathway for our hydrogel may not necessarily require complete molecular degradation in situ, but the physically evacuation via peristalsis. As shown in Figure S14 and Figure 6C,D, the hydrogels were injected on wound sites to form a fully covered layer, such hydrogel layers remained covering on the wound sites on day 5 and day 10 while disappeared on day 14, during which the coverage area decreased as the wounds gradually heals and shrinks. These results indicate the reliability of the hydrogel to serve as the protective layer covered on the wound sites before the full healing of the wounds. The degradation of the PEG block with the molecular weight of 20 kDa (the main component of our hydrogels) has been reported to be ∼3 weeks under physiological conditions [82], which could even retard to months or years by modifying PEG's end hydroxyl groups [82, 83]. Thus, the P(BN‐PEG‐NB)‐based hydrogels, with the NIPAM and BM monomers grafted on PEG end groups, should not be completely degraded on day 14, and the disappear of the hydrogel layers is more likely due to the detachment from the healed wound surfaces. This timely detachment behavior of the hydrogels could ensure the safe and prompt physical clearance of the transportation and excretion of the hydrogel fragments from intestinal tract, avoiding the risks such as infection, inflammation that may be caused by the long‐term retention of the hydrogel as demonstrated in the immunohistochemical analysis (cell proliferation, angiogenesis and inflammatory response) in Figure 6 [84].

3. Conclusion

In conclusion, we have developed an ABA triblock copolymer‐based injectable self‐healing hydrogel as a universal internal wound dressing through the synergy of multiple supramolecular interactions, which is adaptable to diverse wound sites under complex biological conditions. The thermo‐responsive NIPAM units and π‐conjugated BM moieties in terminal “A” blocks were demonstrated to introduce physiological temperature‐enhanced hydrophobic interaction and cation‐induced π‐cation‐π interactions, respectively, serving as reversible cross‐linking interactions within the hydrogel network. Ascribing to the universal adaptability and synergistic effect of these supramolecular interactions across varied biological environments, the as‐formed hydrogel dressing exhibited robust injectability, rapid in situ gelation and reliable self‐healing capacity, unimpaired by environmental complexity. Upon injection into targeted sites in various physiological surroundings, the hydrogel precursor solution could readily undergo sol‐to‐gel transition to form a physical supramolecular hydrogel capable of restoring structure integrity after repeated damage, while the hydrated PEG “B” blocks served as antifouling barriers to inhibit biofoulant adhesion. Gastric and intestinal perforation treatment in in vivo rat model substantiated the broad applicability of the as‐developed hydrogel wound dressing across different physiological situations. Compared with conventional sutures and commercial resorbable barrier films, the hydrogel dressing in this work exhibited advantages in significantly simplifying surgical procedures, reducing postoperative complications and accelerating healing process. This work provides an innovative paradigm for leveraging multiple supramolecular interactions to engineer hydrogel‐based biomaterials that can actively utilize environmental factors (e.g., temperature, pH, salinity) to enhance functionality in complex biological environments, offering broad implications for implantable and interventional biomedical applications beyond gastrointestinal repair and regenerative medicine.

4. Experimental Section

Animal ethics statements: The animal experiments were approved by the Committee on the Ethics of Southeast University (protocol code: 20240829002, date of approval: August 29, 2024).

Materials, experimental methods and procedures, material characterization are available in supporting information.

Author Contributions

Li Xiang: Writing – original draft, Visualization, Investigation, Validation, Methodology, Formal analysis, Data curation, Funding acquisition. Lei Liu: Conceptualization, Supervision, Resources, Project administration, Writing – review & editing. Shixin Zhang: Data curation, Methodology, Investigation, Validation, Visualization. Yuhao Zhang: Data curation, Formal analysis, Validation. Ziqian Zhao: Data curation, Methodology. Jiangwei Chen: Data curation, Methodology, Resource. Jiawen Zhang: Formal analysis, Methodology, Funding acquisition. Jingyi Hu: Data curation, Methodology, Investigation, Resource, Writing – review & editing. Yunfei Chen: Conceptualization, Supervision, Writing – review & editing. Hongbo Zeng: Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review & editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll72806‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.