In situ glycosylation-directed H-aggregation of Type I photosensitizers for synergistic biofilm eradication and promoting diabetic wound healing

Abstract

Biofilm-associated diabetic wound infections pose a major therapeutic challenge. Although photodynamic therapy (PDT) offers an alternative antibacterial strategy, conventional photosensitizers are often limited by inadequate biofilm penetration and poor activity under hypoxic conditions. In this study, we report glycosylated photosensitizers (NBS-Gal and NBS-Lac) that spontaneously form H-aggregates in aqueous media via π-stacking. This self-assembly integrates molecular function with nanostructure without requiring auxiliary components and enables robust Type I photodynamic activity. Notably, NBS-Lac exhibits superior H-aggregation, resulting in 2.2-fold and 1.8-fold higher O₂^˙− generation (after 4 min irradiation) than NBS-NH₂ and NBS-Gal, respectively. The glycosyl moieties enable targeted bacterial recognition through carbohydrate–lectin interactions, while the positive charge on NBS facilitates biofilm penetration via electrostatic interactions. NBS-Lac achieves 100% bactericidal efficacy against P. aeruginosa, along with high biofilm inhibition (∼87%) and eradication (∼80%). In a murine diabetic wound model, NBS-Lac mediates complete healing (100%) under light irradiation, significantly outperforming the controls. This work establishes carbohydrate-directed self-assembly as a novel paradigm for designing targeted, hypoxia-tolerant Type I PDT agents.

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Introduction

Diabetic foot ulcer (DFU) is one of the most serious and common complications of diabetes mellitus, characterized by chronic refractory wounds.¹ These ulcers are highly susceptible to bacterial infection, which severely impedes healing. Improper management of these infected ulcers may lead to amputation. Conventional treatment typically involves the administration of antibiotics. However, the overuse and misuse of antibiotics have led to the emergence of multidrug-resistant bacteria, drastically diminishing their efficacy due to resistance.^2,3 Meanwhile, efforts to develop more potent antibiotics are accelerating the emergence of new superbugs and perpetuating a vicious cycle.^4,5 Alternative therapies like antibacterial photodynamic therapy hold a significant promise for combating bacterial infections while circumventing antibiotic resistance.

A major factor contributing to bacterial resistance is biofilm formation: bacterial communities encapsulated within an extracellular polymeric substance (EPS) matrix.^6,7 This structure not only provides physical protection to bacteria against external threats but also significantly impedes drug penetration. More critically, the hypoxia microenvironment of biofilms limits the application of conventional photosensitizers (Type II PSs).^8–10 Consequently, the development of novel oxygen-independent or minimally oxygen-dependent photosensitizer (Type I PS) system capable of effectively inhibiting and disrupting biofilms has become a pressing imperative for overcoming antimicrobial resistance. Photosensitizers are a key factor for achieving highly efficient aPDA. Enhancing the efficiency of Type-I photosensitizers centers on either blocking energy transfer from Type-II sensitizer excited states to oxygen or improving electron transfer capabilities, thereby shifting the dominant ROS generation pathway from oxygen-dependent Type-II to oxygen-independent Type-I processes.^11–13 However, the design of existing Type-I photosensitizers faces substantial challenges, often necessitating multiple components (e.g., electron-donating and -withdrawing groups), which limits the generality of applicable strategies.^14–20 More importantly, their inadequate targeting capabilities and the aggregation-caused quenching (ACQ) effect induced by π–π stacking in aqueous media severely impair the generation efficiency of reactive oxygen species (particularly superoxide anion, O₂^˙−).^21–25 To address persistent developmental challenges in Type I photosensitizers, efficiency optimization of existing systems represents a more practical alternative.

Sulfur-substituted Nile Blue (NBS)-based photosensitizers have emerged as promising Type I photodynamic therapy (PDT) agents owing to their favorable photophysical properties. However, their therapeutic application is often hindered by suboptimal reactive oxygen species (ROS) generation efficiency and a lack of inherent targeting capability—limitations that are particularly critical in biofilm-associated infections and hypoxic microenvironments. Herein, we present glycosylated Type I photosensitizers based on a NBS scaffold, NBS-Gal and NBS-Lac. The two photosensitizers undergo spontaneous, auxiliary-free self-assembly into π-stacking-driven H-aggregates in aqueous media. This intrinsic supramolecular organization synergistically integrates molecular functionality and nanostructure, dramatically simplifying photosensitizer design. First, glycosylation promotes H-aggregation of the photosensitizers, which facilitates efficient intermolecular charge transfer and significantly elevates reactive oxygen species (ROS) generation. Notably, NBS-Lac demonstrates a greater degree of H-aggregation than both NBS-Gal and the non-glycosylated analogue NBS-NH₂, which corresponds to respective increases of 2.2-fold and 1.8-fold in the quantum yield of O_2˙⁻ (after 4 min irradiation). Second, the photosensizers enable precise bacterial targeting through carbohydrate–lectin interactions with surface glycoproteins. As a result, NBS-Lac achieves ∼100% bactericidal efficacy against P. aeruginosa PA14, alongside high biofilm inhibition (∼87%) and eradication (∼80%) rates. In vivo, NBS-Lac mediates complete healing (∼100%) of infected diabetic wounds under irradiation, surpassing NBS-Gal (∼77%), NBS-NH₂(∼73%) and PBS controls (∼45%). Therefore, the molecular design establishes a carbohydrate-mediated supramolecular assembly mechanism that enables the integration of molecular recognition and photodynamic function within a single nanostructured system. By exploiting carbohydrate-directed H-aggregation, we demonstrate a strategy to concurrently enhance excited-state charge separation and target-specific accumulation, thereby providing a generalizable approach for constructing high-performance, hypoxia-tolerant Type I photosensitizers (Scheme 1).

Scheme 1 Schematic illustration of glycosylation-directed H-aggregation assembly of a Type I photosensitizer into functional nanoconstructs. The resulting NBS-Lac system exhibits over 2.2-fold enhancement in superoxide anion (O₂^˙−) production compared to the parent photosensitizer NBS, enabled by efficient charge separation within H-aggregates for potent antibacterial photodynamic therapy (aPDT).

Results and discussion

Molecular synthesis

The molecular design features a NBS core covalently linked to galactose (NBS-Gal) or lactose (NBS-Lac) motifs. This architecture enables spontaneous H-aggregation in aqueous media, synergistically coupling the intrinsic Type I photochemistry of the NBS core with carbohydrate-driven supramolecular assembly and active targeting. The glycosylated derivatives were synthesized via carboxyl-directed postmodification following established protocols,^26,27 and their structures were fully characterized by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS) (Fig. S20–34).

Self-assembly mechanisms and nanostructural characterization

Self-assembly pathways of NBS-Lac, NBS-Gal, and NBS-NH₂ were elucidated through solvent- and concentration-dependent UV/Vis absorption and fluorescence spectroscopy (Fig. 1a–c). In good solvents (DMSO/CH₃OH), all compounds exhibited a characteristic monomeric absorption band at λ_max ∼660 nm (Fig. 1a–c). These glycosylated derivatives displayed high molar extinction coefficients (ε ≈ 7.9 × 10⁴ M⁻¹ cm⁻¹, 7.4 × 10⁴ M⁻¹ cm⁻¹, and 6.9 × 10⁴ M⁻¹ cm⁻¹), confirming potent light-harvesting capabilities. Incremental water (poor solvent) addition induced progressive attenuation of this monomer peak in NBS-Lac and NBS-Gal, with concomitant emergence of a blue shifted band at 630 nm (Fig. S2). The concentration-dependent amplification of the blue transition unambiguously signifies H-aggregation (distinct from J-aggregate signatures). Notably, H-aggregate formation was exclusive to glycosylated analogues, implicating carbohydrate moieties as critical mediators of supra-molecular ordering. The absorption peak of NBS-Lac at 630 nm is higher than that of NBS-Gal, indicating that lactose-modified NBS is more conducive to H-aggregation. Correlative TEM analyses revealed aqueous self-assembly into monodisperse nanospheres with diameters of ∼100 nm (Fig. 1d and e). This well-defined nanostructure ensures excellent storage stability, retaining 90% of its initial activity after 30 days at 4 °C (Fig. S2d), and is suitable for in vivo application studies. Control experiments with NBS-NH₂ showed neither spectroscopic H-aggregate signatures nor nanoparticle formation, consistent with its inability to undergo carbohydrate-directed assembly.

Fig. 1 (a–c) Absorption spectra of NBS-NH₂, NBS-Gal and NBS-Lac in CH₃OH and H₂O. (d and e) TEM images of NBS-Lac and NBS-Gal. Scale bar: 200 nm. (f) Photodegradation curves of 1,3-diphenylisobenzofuran (DPBF) in the presence of NBS-Lac, NBS-Gal and NBS-NH₂ in CH₂Cl₂ under light irradiation. The experiments were performed in CH₂Cl₂, where all compounds exist as monomers. (g) Maximum fluorescence intensity enhancement of DHR 123 for NBS-NH₂, NBS-Gal and NBS-Lac. (h) Maximum fluorescence intensity enhancement of HPF for NBS-NH₂, NBS-Gal and NBS-Lac. (i) Emission intensity enhancement at 525 nm of SOSG (fluorescent probe for ¹O₂ detection, λ_ex = 504 nm) treated with NBS-NH₂, NBS-Gal and NBS-Lac. The abscissa represents irradiation time. The irradiation was carried out under 660 nm red LED light with a power density of 20 mW cm⁻².

Photodynamic properties of NBS-Lac, NBS-Gal, and NBS-NH₂

To investigate glycosylation-driven H-aggregation effects on ROS generation, we quantitatively evaluated ROS production capabilities of the monomeric and aggregated forms in solution. The singlet oxygen (¹O₂) quantum yields of the monomeric compounds were assessed using DPBF in CH₂Cl₂. Of note, under these conditions, NBS-Lac exists as a monomer (rather than H-aggregates); therefore, the data in Fig. 1f and S3 represent the monomeric state. These monomeric compounds showed no statistically significant differences in ¹O₂ generation. In contrast, aqueous measurements with the SOSG probe revealed that the H-type aggregates formed by NBS-Lac substantially suppress ¹O₂ generation (Fig. 1i and S7), confirming a clear shift from Type II to Type I photodynamic activity. Consistent with this shift, the glycosylated aggregates exhibit markedly enhanced Type I ROS production. NBS-Lac shows a 2.2-fold increase in O₂^˙− generation (after 4 min irradiation) relative to non-glycosylated NBS-NH₂ (Fig. 1g and S4). Hydroxyl radical (^˙OH) production displayed no statistically significant difference among the three compounds (Fig. 1h and S6). ESR measurements with DMPO trapping confirmed O₂˙⁻ photogeneration via characteristic DMPO–O₂^˙− signals (Fig. S5), whereas no ˙OH signals were detected, consistent with probe-based assays. Together, these results establish that H-aggregation in NBS-Lac may promote intermolecular charge separation and electron transfer, thereby selectively boosting Type I ROS generation.

Mechanism of promoting intersystem crossing to enhance ROS generation

To elucidate the regulatory role of glycosylation in governing the aggregation behavior of NBS, a combined molecular dynamics (MD) and theoretical computational study was conducted (Fig. 2a–f).^28,29 MD simulations revealed that glycosylation markedly alters the molecular stacking geometry: The intermolecular distance between neighboring NBS-NH₂ units was 3.19 Å, characteristic of π–π stacking, with a slip angle of ∼39.57°(Fig. 2d), below the critical threshold of 54.7,³⁰ consistent with J-aggregation and aligning with spectral observations. In contrast, NBS-Gal dimers exhibited a slip angle of ∼54.03°(Fig. 2e), suggesting coexisting J- and H-aggregation, whereas NBS-Lac displayed a slip angle of ∼59.27° (>54.7°) with nearly parallel molecular planes (3.49 Å spacing), confirming predominant H-type packing (Fig. 2f). Enhanced face-to-face stacking in NBS-Lac supports its stronger H-aggregation tendency, corroborating the blue-shifted absorption near 620 nm. This carbohydrate-induced face-to-face parallel alignment establishes an optimal platform for enhanced π–π interactions and efficient intermolecular charge transfer.

Fig. 2 Mechanism of enhancing ROS generation through promoting the intersystem crossing (ISC). (a–c) Snapshots of NBS-NH₂, NBS-Gal and NBS-Lac aggregation in water obtained by MD simulation at 298 K. (d–f) Front-view representations depicting the molecular stacking arrangement within the NBS-NH₂, NBS-Gal and NBS-Lac aggregates. The angle (θ) between the transition dipoles and the interconnected axis was measured. Generally, θ < 54.7° favors J-type aggregation, whereas θ > 54.7° favors H-type aggregation. (g and h) Schematic representation of TD-DFT-calculated energy levels, isosurfaces, and key orbital configurations for the singlet and triplet states of NBS and its coplanar dimer.

To gain mechanistic insight into the structure–photofunction relationship, time-dependent density functional theory (TD-DFT) calculations were performed on monomeric and dimeric NBS systems in both singlet and triplet states (Fig. 2g and h). For the NBS monomer, the lowest singlet excited state (S₁, 1.86 eV) is energetically close to the T₂ state (1.14 eV), with a small energy gap (ΔE_ST = 0.12 eV) that facilitates efficient intersystem crossing (ISC). However, T₂ is a charge-transfer (CT) state favorable for electron transfer and radical generation. Rapid internal conversion from T₂ to T₁ subsequently populates a locally excited (LE) T₁ state with an energy >0.98 eV, which preferentially generates ¹O₂ via the Type II pathway. In contrast, the NBS dimer exhibits a smaller energy gap between its lowest singlet (S₁, 1.31 eV) and triplet (T₁, 1.02 eV) states (ΔE_ST = 0.29 eV). For the dimer, the S₁ state is characterized as a typical electron-separated state, with holes and electrons localized on separate monomer units. The triplet excited state features mixed electron-transfer (ET) and charge-separated (CS) configurations, resulting in asymmetric distribution of hole and electron densities across the two monomers. This enhanced electron separation stabilizes the excited state and facilitates electron transfer, thereby boosting ROS generation. Collectively, H-aggregation promotes Type I photodynamic activity through two distinct mechanisms compared to the monomer: (i) shifting the primary ISC pathway from S₁ to T₂ (monomer) to S₁ to T₁ (dimer); and (ii) establishing an electron-separated S₁ state and a mixed ET/CS T₁ state with enhanced electron separation, which favors electron transfer to oxygen for O₂˙⁻ generation. These findings underscore the pivotal role of glycosylation-directed aggregation in tuning photophysical behavior and optimizing photodynamic performance, providing a mechanistic foundation for the development of next-generation hypoxia-active phototherapeutic agents.

In vitro antibacterial PDT

Capitalizing on the capacity of glycosylated-NBS derivatives to generate cytotoxic ˙OH and O₂˙⁻, we evaluated their photodynamic antibacterial efficacy against Pseudomonas aeruginosa PA14, a clinically relevant pathogen in diabetic wound infections. Prior to assessing antibacterial performance, we evaluated the biocompatibility of the compounds. Using MTT assays with L929 murine fibroblasts, we observed >90% cell viability after 24 h incubation with 32 µM photosensitizer, demonstrating minimal dark toxicity (Fig. S18). Hemocompatibility studies further showed less than 5% hemolysis in 4% murine erythrocyte suspensions treated with 32 µM compound (4 °C, 1 h), compared to Triton X-100 controls, confirming high biocompatibility under dark conditions for in vivo applications (Fig. S19). Using spread plate assays under both normoxic (21% O₂) and hypoxic (1% O₂) conditions, we observed negligible dark toxicity (>90% viability, Fig. S8a, b, S9a and b). NBS-Lac mediated PDT achieved complete bacterial eradication (100% kill rate) at 2.8 µM under normoxia, outperforming NBS-Gal and NBS-NH₂, which retained ∼26% and ∼41% viable cells, respectively (Fig. 3b and c). Remarkably, under hypoxic conditions—where conventional photosensitizers often fail—NBS-Lac maintained complete bactericidal activity, whereas NBS-Gal and NBS-NH₂ showed substantially reduced efficacy, with survival rates of ∼33% and ∼52%, respectively (Fig. 3d and e). Live/dead staining and SEM further validated these results: SYTO9/PI staining showed near-total bacterial death (red fluorescence) for NBS-Lac, contrasting with residual live cells (green) in NBS-Gal/NBS-NH₂ groups (Fig. 3f). SEM revealed severe NBS-Lac-induced morphological disruption, including cell collapse and fragmentation after irradiation, exceeding damage caused by the other derivatives (Fig. 3g). To confirm the specificity of carbohydrate–lectin-mediated bacterial targeting, a competitive inhibition assay was performed using free lactose as a blocking agent. As shown in Figure S10, Pseudomonas aeruginosa PA14 uptake of NBS-Lac and NBS-Gal was significantly reduced—by approximately ∼48% and ∼27%, respectively—in the presence of excess free lactose, whereas no inhibition was observed for the non-glycosylated control (NBS–NH₂). These results confirm that the targeting is indeed mediated by specific carbohydrate–lectin interactions. The observed bactericidal activity is likely driven by both glycan-mediated bacterial recognition and lactose-enhanced H-aggregation, enabling oxygen-independent photodynamic activity that is particularly well suited for hypoxic wound therapy.

Fig. 3 (a) In vitro antibacterial activities of NBS-NH₂, NBS-Gal and NBS-Lac series compounds against planktonic P.A. (b) and (d) Images of P.A. colonies on LB agar plates after light treatments in normoxia and hypoxia. (c) and (e) Quantitative analysis of the bacterial cell viability based on the results of colony counting. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. (f) Live/dead staining of bacteria after incubation with synthetic probes. Confocal images of PA after different treatments (for SYTO9 green live bacterial stain, λ_ex = 488 nm and λ_em = 500–550 nm; for PI red dead bacterial stain, λ_ex = 561 nm and λ_em = 571–630 nm), scale bar: 10 µm. (g) Representative SEM images of P.A. stained by NBS-Lac, NBS-Gal and NBS-NH₂ after different treatments, respectively. Scale bar: 1 µm.

Anti-biofilm activity of NBS-Lac, NBS-Gal, and NBS-NH₂

The oxygen-independent bactericidal efficacy of NBS-Lac highlights the therapeutic advantage of this glycosylated photosensitizer in combating hypoxic wound infections. Building on its potency against planktonic P. aeruginosa, we evaluated NBS-Lac's capacity to combat P. aeruginosa biofilms—notorious for thriving in anaerobic niches and impeding treatment. The ability of NBS-Lac, NBS-Gal, and NBS-NH₂ to inhibit the formation of P. aeruginosa biofilms was first evaluated and quantified by crystal violet staining. Crystal violet quantification revealed that NBS-Lac (2.1 µM, 24 h incubation in the dark) reduced biofilm formation by ∼88%, significantly surpassing NBS-Gal (∼75%) and NBS-NH₂ (∼66%) (Fig. 4b). Live/dead staining corroborated this superior biofilm–inhibitory activity (Fig. 4d). Critically, the absence of illumination implicates lactose-mediated bacterial adhesion blockade as the mechanism driving biofilm suppression rather than ROS generation. Disrupting mature biofilms poses a substantially greater challenge than inhibiting their formation, primarily due to the dense extracellular polymeric matrix that severely restricts photosensitizer penetration. To assess the anti-biofilm potential of NBS-Lac, NBS-Gal and NBS-NH₂ against pre-established P. aeruginosa biofilms, we administered treatments under light conditions. At 9.6 µM, NBS-Lac demonstrated remarkable efficacy in disrupting mature biofilms (∼80% eradication), far exceeding the moderate effects observed with NBS-Gal (67%) and NBS-NH₂ (56%) (Fig. 4c). Consistent with these results, Live/dead fluorescence assays confirmed the potent bactericidal effect of NBS-Lac (Fig. 4e and S11). In addition, NBS-Lac treatment produced significantly thinner biofilms than those observed in the NBS-Gal and NBS-NH₂ groups. The superior disruptive efficacy is attributed to a dual-mechanism synergy: (1) cationic charge-enhanced diffusion through the biofilm matrix, and (2) glycosylation prompted the formation of H-aggregates, which significantly boosted the efficiency of ROS generation. The elevated ROS levels then degraded the extracellular polymeric matrix, enabling enhanced photosensitizer permeation and ultimately positioning it as an integrated therapeutic strategy for treating biofilm-infected hypoxic wounds.

Fig. 4 The antibacterial application and the ability of NBS-NH₂, NBS-Gal and NBS-Lac to inhibit/disrupt biofilm formation. (a) Schematic illustration of the mechanism of NBS-NH₂, NBS-Gal and NBS-Lac for inhibiting the formation of bacterial biofilms. (b) Inhibition percentage of PA14 biofilm formation with various concentrations of NBS-NH₂, NBS-Gal and NBS-Lac. (c) Disruption percentage of PA14 biofilm formation with various concentrations of NBS-NH₂, NBS-Gal and NBS-Lac. (d) CLSM images of NBS-NH₂, NBS-Gal and NBS-Lac for inhibiting the formation of PA14 biofilms. Scale bar: 20 µm. (e) CLSM images of NBS-NH₂, NBS-Gal and NBS-Lac for disrupting the formation of PA14 biofilms. Scale bar: 20 µm.

In vivo antibacterial activity of NBS-Lac, NBS-Gal, and NBS-NH₂

Diabetic wounds have a heightened vulnerability to P. aeruginosa colonization and infection, significantly exacerbating the difficulty of effective treatment and presenting greater challenges for treatment control.³¹ Motivated by promising in vitro antibacterial activity and biofilm inhibition/clearance capabilities of NBS-Lac, we evaluated the therapeutic efficacy of NBS-Lac in a P. aeruginosa infected diabetic rat wound model. Animals were randomized into eight groups: (1) PBS, (2) PBS + light, (3) NBS-NH₂, (4) NBS-NH₂ + light, (5) NBS-Gal, (6) NBS-Gal + light, (7) NBS-Lac, and (8) NBS-Lac + light; Additionally, three healthy rats without wound induction served as negative controls. Wound healing was monitored every two days via photographic documentation (closure rates) and bacterial burden analysis, alongside body weight measurements to assess systemic effects. As shown in Fig. 5b–f, the NBS-Lac + light group exhibited the highest healing rate, achieving complete wound closure by day 8, whereas ∼57% of wounds in the PBS group remained unhealed. Notably, NBS-Lac achieved ∼63% wound closure, suggesting that NBS-Lac can inhibit P. aeruginosa biofilm formation (Fig. S12 and S13). These findings are consistent with the results of the in vitro biofilm inhibition and clearance assays. More importantly, bacterial plate counts from infected tissue homogenates confirmed near-complete bacterial eradication in the NBS-Lac + light group, underscoring its potent in vivo photodynamic antibacterial activity (Fig. 5d, g and S14).

Fig. 5 The ability of NBS-NH₂, NBS-Gal and NBS-Lac to promote wound healing in PA14-infected rats in vivo. (a) Scheme of the therapeutic process of PA14-infected diabetic wound healing. (b) Representative images of wound change in diabetic rats infected with PA14 after different treatments. Scale bar: 4 mm. (c) Traces of wound-bed closure in diabetic rats infected with PA14 after different treatments. (d) Representative images of bacteria colonies remaining in wounds of diabetic rats infected with PA14 after different treatments. (e) Wound area changes in PA14 infected diabetic rats after different treatments. (f) Wound healing rates in PA14 infected diabetic rats after treatment. (g) Remaining bacterial counts in MDR PA infected diabetic rats after different treatments. *p < 0.05, **p < 0.01, and ***p < 0.001.

Histological analysis on day 8 post-treatment provided further evidence of the superior wound healing efficacy (Fig. 6). The NBS-Lac + light group exhibited the most advanced tissue regeneration, characterized by the thickest granulation tissue (∼99 µm), a value substantially exceeding those observed in all other treatment groups, a finding further evidenced by the highest collagen deposition (∼62%), approaching that of healthy skin (∼65%) (Fig. 6a–c and S15a). These regenerative effects were accompanied by robust angiogenesis, as evidenced by the highest CD31 expression among all groups (Fig. 6g, h and S15c). Critically, NBS-Lac + light treatment effectively resolved inflammation, reducing TNF-α and IL-6 expression to levels comparable to those in healthy controls (2–3%), whereas PBS and non-irradiated groups exhibited persistent inflammatory responses indicative of uncontrolled infection (Fig. 6d–f and S15b). Collectively, these findings demonstrate that NBS-Lac upon 660 nm irradiation not only exerts potent antibacterial activity but also accelerates diabetic wound repair through a coordinated mechanism involving enhanced tissue regeneration, collagen deposition, inflammation suppression, and angiogenesis. In vivo bio-safety was confirmed by normal body weight gain (Fig. S16) and the absence of histopathological abnormalities in major organs (Fig. S17).

Fig. 6 (a) H&E and Masson staining of wound regeneration after various treatments on day 8. (b and c) Quantitative analysis of granulation tissue thickness and collagen deposition on day 8 post-treatment. (d) Immunohistochemical staining of TNF-α and IL-6. (e and f) Quantitative analysis of TNF-α and IL-6 in wound tissues on day 8. (g) Immunofluorescence images of CD31 staining. Scale bar was 100 nm. (h) Quantitative analysis of CD31 in wound tissues on day 8. Data are presented as mean ± SD, n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001.

Conclusion

In summary, we have designed and synthesized two novel glycosylated Type I photosensitizers, NBS-Lac and NBS-Gal, which integrate bacterial targeting, enhanced O₂˙⁻ generation, and hypoxia tolerance within a single molecular platform. These molecules spontaneously self-assemble into H-aggregates in aqueous media via π-stacking, a supramolecular organization that promotes intermolecular charge transfer and facilitates intersystem crossing, leading to markedly elevated O₂˙⁻ production. Notably, NBS-Lac exhibits 2.2-fold higher O₂˙⁻ production (after 4 min irradiation) than its non-glycosylated counterpart, NBS-NH₂. This enhanced ROS generation translates to potent antibacterial activity under both normoxic and hypoxic conditions, effective eradication of drug-resistant P. aeruginosa biofilms, and accelerated healing of infected diabetic wounds in vivo. Comprehensive safety evaluations further underscore the translational potential of this molecular design for combating hypoxic biofilm-associated infections.

Author contributions

D. X.: design, photophysical studies, data analysis, writing – original draft, formal analysis, supervision, and funding acquisition. W. J. synthesized and characterized the fluorescent probe. M. L. designed and conducted the biological experiments and biological assays. J. G. and P. W. conducted immunohistochemical staining and analysis. Q. Y., X. F. and X. P.: validation, data analysis and review. K. W. and W. S.: methodology, formal analysis, writing, review, supervision, and funding acquisition. All co-authors wrote and edited the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

All experimental procedures and associated data are provided in the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6sc03993f.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (22408080), the Natural Science Foundation of Hebei Province (B2023201080), the State Key Laboratory of Fine Chemicals, Dalian University of Technology (KF2309), and the Foundation of Hebei University (050001-521100222046) for financial support.

References

1. J. Li, Y. Hu and B. Hu, et al., Nat. Commun., 2022, 13, 627.
2. M. Baym, L. K. Stone and R. Kishony, et al., Science, 2015, 325, aad3292.
3. E. Brown and G. Wright, Nature, 2016, 529, 336.
4. P. Klenotic, M. Moseng and C. Morgan, et al., Chem. Rev., 2021, 121, 5378.
5. P. He, M. Jia and L. Yang, et al., Adv. Mater., 2025, 37, 2418978.
6. D. Hu, H. Li and B. Wang, et al., ACS Nano, 2017, 11, 9330.
7. C. R. Arciola, D. Campoccia and L. Montanaro, Nat. Rev. Microbiol., 2018, 16, 397.
8. F. Guo, J. Yoo and Q. Zhang, et al., J. Am. Chem. Soc., 2025, 147, 27932.
9. W. Chen, Y. Zhang and H. Yi, et al., Angew. Chem., Int. Ed., 2023, 62, e202300162.
10. T. Xiong, F. Ning and Y. Chen, et al., ACS Nano, 2025, 19, 2822.
11. K. Teng, L. Niu and J. Li, et al., Angew. Chem., Int. Ed., 2025, 64, e202509416.
12. V. Lechner, M. Nappi and P. Deneny, et al., Chem. Rev., 2022, 122, 1752.
13. L. Zhu, X. Wang and T. Tian, et al., Chem. Sci., 2024, 15, 10499.
14. K. X. Teng, D. Zhang and B. K. Liu, et al., Angew. Chem., Int. Ed., 2024, 63, e202318783.
15. M. Li, Y. Xu and Z. Pu, et al., Proc. Natl. Acad. Sci. U. S. A., 2022, 119, 2210504119.
16. K. X. Teng, L. Y. Niu and Q. Yang, J. Am. Chem. Soc., 2023, 145, 4081.
17. T. Xiong, Y. Chen and Q. Peng, et al., Adv. Mater., 2024, 36, 2309711.
18. J. Zhuang, G. Qi and Y. Feng, et al., Nat. Commun., 2024, 15, 4943.
19. X. Li, D. Lee and J.-D. Huang, et al., Angew. Chem., Int. Ed., 2018, 57, 9885.
20. X. Lan, H. Li and Y. Liu, et al., Angew. Chem., Int. Ed., 2024, 63, e202407092.
21. J. Liu, D. W. Kang and Y. Fan, et al., J. Am. Chem. Soc., 2024, 146, 849.
22. M. Li, J. Xia and R. Tian, et al., J. Am. Chem. Soc., 2018, 140, 14851.
23. Z. An, C. Zheng and Y. Tao, et al., Nat. Mater., 2015, 14, 685.
24. T. J. Penfold, E. Gindensperger and C. Daniel, et al., Chem. Rev., 2018, 118, 6975.
25. Z. Wang, W. Ma and Z. Yang, et al., J. Am. Chem. Soc., 2024, 146, 28973.
26. Y. Chen, T. Xiong and Q. Peng, et al, Nat. Commun., 2024, 15, 6935.
27. J. Yang, F. Yin and H. Li, et al, Adv. Healthcare Mater., 2025, 14, 2404859.
28. D. Xi, M. Xiao and J. Cao, et al, Adv. Mater., 2020, 32, 1907855.
29. D. Xi, N. Xu and X. Xia, et al, Adv. Mater., 2022, 34, 2106797.
30. C. Sun, B. Li and M. Zhao, et al, J. Am. Chem. Soc., 2019, 141, 19221.
31. J. Makabenta, A. Nabawy and C. Li, et al, Nat. Rev. Microbiol., 2021, 19, 23.

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