Abstract

   With the threat posed by drug‐resistant pathogenic bacteria, developing
   non‐antibiotic strategies for eradicating clinically prevalent
   superbugs remains challenging. Ferroptosis is a newly discovered form
   of regulated cell death that can overcome drug resistance. Emerging
   evidence shows the potential of triggering ferroptosis‐like for
   antibacterial therapy, but the direct delivery of iron species is
   inefficient and may cause detrimental effects. Herein, an effective
   strategy to induce bacterial nonferrous ferroptosis‐like by
   coordinating single‐atom metal sites (e.g., Ir and Ru) into the
   sp^2‐carbon‐linked covalent organic framework (sp^2c‐COF‐Ir‐ppy[2] and
   sp^2c‐COF‐Ru‐bpy[2]) is reported. Upon activating by light irradiation
   or hydrogen peroxide, the as‐constructed Ir and Ru single‐atom
   catalysts (SACs) can significantly expedite intracellular reactive
   oxygen species burst, enhance glutathione depletion‐related glutathione
   peroxidase 4 deactivation, and disturb the nitrogen and respiratory
   metabolisms, leading to lipid peroxidation‐driven ferroptotic damage.
   Both SAC inducers show potent antibacterial activity against
   Gram‐positive bacteria, Gram‐negative bacteria, clinically isolated
   methicillin‐resistant Staphylococcus aureus (MRSA), and biofilms, as
   well as excellent biocompatibility and strong therapeutic and
   preventive potential in MRSA‐infected wounds and abscesses. This
   delicate nonferrous ferroptosis‐like strategy may open up new insights
   into the therapy of drug‐resistant pathogen infection.

   Keywords: anti‐infection therapy, covalent organic frameworks,
   ferroptosis‐like, single‐atom catalysts, transition metal complex
     __________________________________________________________________

   A novel nonferrous ferroptosis‐like strategy by coordinating
   single‐atom metal sites (e.g., Ir and Ru) into the sp^2‐carbon‐linked
   covalent organic framework is reported. The as‐constructed Ir and Ru
   sites can effectively induce lipid peroxidation‐driven bacterial
   ferroptotic damage, showing strong therapeutic and preventive potential
   in infected wounds and abscesses.

   graphic file with name ADVS-10-2207507-g005.jpg

1. Introduction

   Antimicrobial resistance severely compromises traditional chemotherapy
   regimens and persists as a worldwide health problem.^[ [42]^1 ^]
   Bacteria can employ a variety of strategies to avert growth suppression
   of conventional antibiotic therapy, consisting of enzyme inactivation
   and target modification.^[ [43]^2 ^] Furthermore, they could spark
   post‐antibiotic dilation and recurrent/persistent infection through
   reprogramming host metabolism, interfering with degradation pathways,
   and inhibiting immune cells.^[ [44]^3 ^] Alternative strategies capable
   of circumventing antibiotic resistance are of utmost importance.
   Ferroptosis is an iron‐dependent form of cell death resulting from
   lipid peroxidation (LPO) that has been implicated in various biological
   contexts, from development to aging to immunity and cancer.^[ [45]^4 ^]
   Several types of strategies have been described to induce ferroptosis
   to date, comprising iron delivery,^[ [46]^5 ^] system Xc^−
   suppression,^[ [47]^6 ^] glutathione (GSH) depletion, and glutathione
   peroxidase 4 (GPX4) inhibition.^[ [48]^7 ^] The precise mechanism that
   eventually results in ferroptotic cell death probably involves damage
   to membrane integrity, disruption of membrane properties through lipid
   cross‐linking, and further oxidative impairment to macromolecules and
   cellular structures induced by reactive oxygen species (ROS) derived
   from polyunsaturated fatty acid chains.^[ [49]^8 ^] It has been
   hypothesized that the regulation of key molecules in classical
   regulatory pathways of ferroptosis can serve as a potential approach to
   overcoming drug resistance.^[ [50]^9 ^]

   Emerging ferrous‐based nanomaterials, comprising ferumoxytol, nano‐iron
   sulfides, and iron–organic frameworks, have been applied as inducers
   for ferroptosis benefiting from the Fenton reaction impelled by
   Fe^2+.^[ [51]^10 ^] For instance, attempts have been made to introduce
   iron ions into the metastable Fe[3]S[4] (greigite) or FeSO[4] to
   facilitate the iron overload‐triggered GSH consumption, leading to the
   ferroptosis‐like of bacterial cells.^[ [52]^11 ^] Ferroferric
   oxide‐based nanoassemblies were also utilized as the inducers for
   bacterial ferroptosis‐like death by eliciting intracellular iron
   overload and iron metabolism interference.^[ [53]^12 ^] However, these
   current iron‐based nanomaterials used to induce bacterial
   ferroptosis‐like are far from satisfactory, requiring very high Fe
   doses or supplementary ingredients to achieve combinatorial effects in
   general.^[ [54]^10b ^] Alternatively, the direct delivery of iron
   species may cause detrimental effects such as neurovirulence, oxidative
   stress, and anaphylactic reactions in normal tissues.^[ [55]^13 ^]
   Single‐atom catalysts (SACs) have become an exciting frontier recently
   in chemical catalysis because of their precisely identified active
   centers, robust catalytic performances, and high stability.^[ [56]^14
   ^] SACs can be regarded as the extreme limit of the precise design of
   nanocatalytic materials at the atomic level. Particularly, they have
   been utilized as bio‐inspired nanozymes to mimic natural enzymes’
   structure and excellent catalytic ability, efficiently generating
   excessive ROS for bacterial or tumor inhibition.^[ [57]^15 ^] For
   example, Qu and colleagues report a self‐adapting iron‐based SAC to
   accelerate selective and safe ferroptosis;^[ [58]^16 ^] a
   nonferrous‐based Pd‐SAC with simulated activities of double peroxidase
   (POD) and glutathione oxidase (GSHOx) also efficiently induces
   ferroptosis characterized by upregulation of LPO and ROS.^[ [59]^17 ^]
   Unfortunately, considering the low intracellular H[2]O[2] level of
   bacterial cells, Fenton reaction alone is difficult to generate
   sufficient ROS, which weakens the catalytic therapeutic efficacy of
   conventional SACs.^[ [60]^18 ^]

   Recently, isolated active metal centers anchored to solid support
   represent an innovative breakthrough in photochemistry.^[ [61]^19 ^]
   Nanoscale covalent organic frameworks (COFs) composed of suitable
   building blocks and organic functional groups have emerged as highly
   promising carriers for their tunable microstructures and optically
   electrical properties in preference to traditional catalyst supports.^[
   [62]^20 ^] The possibility of utilizing COFs as supporting materials to
   construct SACs to meet the requirements of ferroptosis was
   investigated. Various monatomic metal centers anchored on COFs could
   present effective photocatalysis.^[ [63]^21 ^] Many studies have
   certified that transition‐metal elements such as Ir and Ru could act as
   single‐atom active sites without disrupting the framework for
   constructing high‐performance photochemical catalysts.^[ [64]^22 ^]
   Adding single transition metal atoms to the bare photocatalysts can
   expand the optical response range, shorten the electron transfer
   distance, and form stable intermediate configurations in photocatalytic
   reactions by virtue of the increased delocalization effect, enduing
   SACs with excellent photocatalytic performance.^[ [65]^21 , [66]^23 ^]
   Transition metal SACs have also been reported to exhibit POD and GSHOx
   activity.^[ [67]^24 ^] Hence, exploring a COF‐based SAC paradigm for
   bacterial ferroptosis‐inducing agents is imperative and highly
   desirable.

   Herein, we prepare two types of monatomic transition metal sites (e.g.,
   Ir and Ru) anchored on sp^2c‐linked COF (sp^2c‐COF) skeletons with a
   metal–nitrogen–carbon bridging structure (Scheme [68] 1 ). Facilitated
   by covalent interactions in the Schiff base reaction, methoxy
   polyethylene glycol amine (mPEG‐NH[2]‐4000) polymer could be coated to
   produce the hydrophilic and highly biocompatible SACs
   (sp^2c‐COF‐Ir‐ppy[2] and sp^2c‐COF‐Ru‐bpy[2]). The experimental results
   and density functional theory (DFT) calculation indicated that the
   excellent photocatalytic capacity and POD activity of Ir and Ru SACs
   were attributed to the intrinsic porous properties of the COF and the
   synergistic effect between atomically dispersed metal centers and
   sp^2c‐COF hosts. Upon irradiation, the Ir and Ru active sites could
   cause the production of suprathreshold ROS, the consumption of
   intracellular GSH, and the disturbance of respiratory chain and
   metabolism together facilitating irreversible LPO‐driven
   ferroptosis‐like pathways. Both inducers show low hemolysis and
   cytotoxicity, as well as potent antibacterial activity against various
   bacteria, drug‐resistance bacteria, and strong therapeutic and
   preventive potential for methicillin‐resistant Staphylococcus aureus
   (MRSA)‐induced infections in the wound and abscess models.
   Collectively, we conducted a proof‐of‐concept study to discover
   COF‐based SAC as an antibacterial ferroptosis‐like initiator to
   eliminate infections.

Scheme 1.

   Scheme 1
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   Scheme depicting coordinative synthesis of Ir and Ru SACs in the pore
   of sp^2c‐COF.

2. Results and Discussion

2.1. Synthesis and Characterization

   Substantial attention has recently been focused on the olefin‐based
   COFs with fully π‐conjugated systems, a new class of promising
   semiconductor materials.^[ [70]^25 ^] The robust C=C bond not only
   endues the framework with excellent stability under harsh conditions
   but also ensures efficient electron transfer via extensive
   π‐conjugation throughout the framework.^[ [71]^26 ^] Nevertheless, due
   to the poor reversibility of C=C bond formation, the preparation of
   sp^2‐carbon conjugated COFs maintains a tremendous challenge.
   Fortunately, under solvothermal conditions (tetrahydrofuran/0.1 m
   Cs[2]CO[3] = 20/1 v/v, 3 days, 120 °C), the topology‐directed
   polycondensation of C [3]‐symmetric
   4,4′,4″‐(1,3,5‐triazine‐2,4,6‐triyl)tribenzaldehyde (TA) as a knote and
   C [2]‐symmetric linear 2,6‐dicyanomethylbenzo[1,2‐d:4,5‐d′]bisthiazole
   (BTHAN) as a linker yielded a sp^2c‐COF (Figures [72]S1–S4, Supporting
   Information). The establishment of a donor–acceptor (D–A) conjugated
   system in COF by covalent linking of sulfur‐containing aromatic
   heterocyclic sites with triazine active sites would be an effective
   strategy to enhance intramolecular charge transfer capability to
   promote π‐electron delocalization, resulting in a narrower bandgap and
   desirable highest occupied molecular orbital (HOMO) and lowest
   unoccupied molecular orbital (LUMO) energy levels.^[ [73]^27 ^] This
   push–pull effect was demonstrated by calculating the molecular orbital
   density of the ligand by Gaussian at the B3LYP/6‐31G* theoretical level
   (Figure [74] 1a). The HOMO orbitals of BTHAN are mainly concentrated on
   the benzothiazole portion, while the LUMO is delocalized mainly over
   the triazine units, suggesting a much higher degree of LUMO–HOMO
   separation, leading to more efficient charge separation.

Figure 1.

   Figure 1
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   Characterizations of sp^2c‐COF. a) HOMO and LUMO of BTHAN and TA. b)
   Extended structures of non‐interpenetrated sp^2c‐COF. c) Electrostatic
   potential surface of sp^2c‐COF repetitive units showing possible active
   sites. d) The charge density differences of sp^2c‐COF and its magnified
   top and side views, with magenta and yellow representing electron
   accumulation and depletion, respectively. e) Hole and electron
   distribution heat map in the excited state of sp^2c‐COF fragment. f)
   The direction of the dipole moment produced by the local polarization
   of sp^2c‐COF. g) The electron–hole distributions of sp^2c‐COF in the
   excited state, with blue and green representing electron accumulation
   and depletion, respectively. h) The Pawley refinement of sp^2c‐COF: the
   experimental XRD pattern is shown in black, the Pawley refinement
   pattern in red, their difference in blue, and the simulated pattern
   using AA stacking mode in green. i) Crystal structures of the sp^2c‐COF
   AA stacking model. j) N[2] adsorption–desorption isotherms for
   sp^2c‐COF at 77 K. Inset: pore‐size distribution calculated by fitting
   the NLDFT model to adsorption data. k) The simulation pore size of
   sp^2c‐COF in AA stacking. l,m) AFM image and its 3D topography image of
   sp^2c‐COF. n,o) Surface potential image and its 3D potential lines
   profile of sp^2c‐COF.

   For the observation and comparison of local polarization and charge
   separation behavior of the synthesized COF molecules, we first
   exploited DFT to study the optimized molecular structures
   (Figure [76]1b,c). Noticeably, the electrostatic potential analysis for
   sp^2c‐COF exhibits a positive distribution mainly on TA units, while
   the negative regions are mainly located on the cyanide group of the
   BTHAN portion, indicating that the positive and negative charges were
   obviously separated due to the effect of polarization on the local
   charge density triggered by the D–A characteristics of the backbones.^[
   [77]^28 ^] Afterward, the charge separation ability of COFs in ground
   state and excited state was investigated. First, the benzothiazole
   group was recognized as an electron accumulator of sp^2c‐COF in the
   ground‐state charge density differences (Figure [78]1d), yet the
   electrons in the TA group were depleted, indicating an uneven charge
   distribution. Thereafter, BTHAN can create more holes and electrons
   according to the heat map for sp^2c‐COF fragments (Figure [79]1e). In
   addition, the dipole moment of the TA to BTHAN molecule was calculated
   as 17.98 Debye, demonstrating the presence of local charge polarization
   (Figure [80]1f). The excited‐state charge‐separation behavior of
   sp^2c‐COF was analyzed with the electron–hole distribution, in which an
   obvious spatial charge separation was examined (Figure [81]1g). To sum
   up, these results demonstrate that the local polarization in a single
   sp^2c‐COF molecule can lead to significant charge separation.

   The powder X‐ray diffraction (PXRD) measurement exhibits three distinct
   peaks at 2.390, 4.633, and 21.817, indexed as the (100), (200), and
   (001) reflections, respectively (Figure [82]1h, black curve). The
   optimization for the 2D monolayer conformation and configuration of
   different stacking models has been conducted via the density
   function‐based tight binding (DFTB) method. The AA‐stacking model with
   the most favorable energy was obtained and the yielded PXRD pattern
   (Figure [83]1h, green curve) is consistent with the profile examined in
   the experiment. The Pawley‐refined PXRD pattern (Figure [84]1h, red
   curve) with the space group P6/m and unit cell parameters of a = b =
   44.6378 Å, c = 3.5311 Å, and c/a = 0.0791 reproduced the curve observed
   in the experiment with negligible differences (Figure [85]1h, blue
   curve). Table [86]S1 of the Supporting Information summarizes atomic
   atomistic coordinates generated by DFTB calculation and Pawley
   refinement, respectively. Hence, the reconstructed sp^2c‐COF reveals an
   extended hexagonal 2D lattice with sp^2 carbon skeleton along the x and
   y directions (Figure [87]1i). The existence of the (001) plane at
   21.817° indicates the structural order of 3.5 Å separation in the z
   direction perpendicular to the 2D sheet.

   Fourier transform infrared (FT IR) spectroscopy disclosed that for both
   BTHAN monomer and sp^2c‐COF, the cyano group exhibits a stretching
   vibrational peak at 2248 cm^−1 (Figure [88]S5, Supporting Information).
   A peak at 1704 cm^−1 ascribed to the C=O stretching vibration was
   observed in monomer TA and was widely attenuated in sp^2c‐COF,
   suggesting that there was a high degree of polymerization in the
   skeleton. The newly formed peak at 3047 cm^−1 in sp^2c‐COF can be
   attributed to CH=C stretching, which clearly indicated the C=C
   connection in the skeleton. Solid‐state ^13C cross‐polarization
   magic‐angle spinning nuclear magnetic resonance (^13C CP‐MAS NMR)
   spectroscopy showed a peak at 163 ppm, confirming the presence of a
   thiazole ring (Figure [89]S6, Supporting Information). Peaks of ≈105
   and 115 ppm further supported the formation of vinylene bonds and the
   presence of cyano units.^[ [90]^29 ^] These remarkable features
   manifested the successful condensation of the monomers. Nitrogen
   adsorption–desorption tests were carried out at 77 K and the
   characteristic type I curves have been recognized in the simulated
   shape diagram (Figure [91]1j). At low relative pressure, gas absorption
   increases sharply (P/P [0] < 0.1), which indicates the presence of
   micropores. The Brunauer–Emmett–Teller surface area was calculated to
   be 209.5 m^2 g^−1. The average aperture obtained by DFT fitting is
   ≈4.2673 nm, which corresponds to the theoretical value (4.8835 nm) in
   AA models (Figure [92]1k). AFM displayed that the surface roughness of
   sp^2c‐COF is around tens of nanometers (Figure [93]1l,m). The
   significant difference in the surface potential of the sp^2c‐COF layer
   in vertical‐contact mode demonstrates remarkable local
   polarization‐induced charge separation characteristics
   (Figure [94]1n,o).^[ [95]^30 ^] Finally, thermogravimetric analysis
   (TGA) suggested that sp^2c‐COF performs excellent thermal stability,
   even with a residual carbon content of more than 50% up to 800 °C
   (Figure [96]S7, Supporting Information).

   Postsynthetic metalation of sp^2c‐COF involved dispersing it in
   dichloromethane or dichloromethane/methanol in the presence of dimer
   [Ir[2](ppy)[4]Cl[2]] and dimer [Ru[2](bpy)[2]Cl[2]] (Figure [97] 2a).
   For this metalation, dimeric transition complexes were chosen as
   iridium and ruthenium sources since they are capable of binding
   powerful photoredox complexes through coordination with the thiazole
   and cyanogen ligands present in sp^2c‐COF.^[ [98]^31 ^] The content of
   Ir and Ru incorporated into the metalized sp^2c‐COF
   (sp^2c‐COF‐Ir‐ppy[2]and sp^2c‐COF‐Ru‐bpy[2]) was analyzed with an
   inductively coupled plasma optical emission spectrometer (ICP‐OES). The
   contents of Ir and Ru in COFs were 5.37 and 2.12 wt%, respectively.
   These values imply that 31.6% of the thiazole and cyan ligands in
   sp^2c‐COF are coordinated with Ir and 23.7% with Ru. Interestingly,
   metal absorption in the solution is quite efficient. In reality, 50% of
   the Ir species in solution are bound to the material, whereas 22% of
   the Ru precursors are bound to the framework. In addition, FT IR, ^13C
   CP‐MAS NMR, and TGA for sp^2c‐COF‐Ir‐ppy[2] and sp^2c‐COF‐Ru‐bpy[2]
   showed no significant differences between the original and metalized
   COFs (see the Supporting Information). Note that the zeta potential of
   sp^2c‐COF increased positively from −28.1 to −10.2 and +16.8 mV, after
   postsynthetic metalation of Ir and Ru (Figure [99]S8, Supporting
   Information). The stability of sp^2c‐COF‐Ir‐ppy[2] and
   sp^2c‐COF‐Ru‐bpy[2] was also proven in neutral and weakly acidic
   physiological mediums, such as PBS (0.1 m, pH 7.4) and PBS (0.1 m, pH
   6.0). As demonstrated in Figure [100]S9 of the Supporting Information,
   both samples demonstrated superior dispersibility and stability in a
   given medium for over one week without forming any significant
   aggregations. Furthermore, the metal‐centered atoms in
   sp^2c‐COF‐Ir‐ppy[2] and sp^2c‐COF‐Ru‐bpy[2] were stable and negligible
   Ir or Ru‐release was observed for one‐week incubation in both neutral
   and weakly acidic conditions (Figure [101]S9, Supporting Information).

Figure 2.

   Figure 2
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   Morphologies of Ir and Ru SACs. a) Synthetic route of
   sp^2c‐COF‐Ir‐ppy[2] and sp^2c‐COF‐Ru‐bpy[2]. b–d) FESEM images of b)
   sp^2c‐COF, c) sp^2c‐COF‐Ir‐ppy[2], d) and sp^2c‐COF‐Ru‐bpy[2]. e–g) TEM
   and HRTEM images of e) sp^2c‐COF, f) sp^2c‐COF‐Ir‐ppy[2], g) and
   sp^2c‐COF‐Ru‐bpy[2]. h–j) TEM‐EDX mapping images of C, N, Ir, and Ru
   elements in selected areas of h) sp^2c‐COF, i) sp^2c‐COF‐Ir‐ppy[2], j)
   and sp^2c‐COF‐Ru‐bpy[2] (dark field mode). k–m) SAC‐HAADF‐STEM images
   of k) sp^2c‐COF, l) sp^2c‐COF‐Ir‐ppy[2], and m) sp^2c‐COF‐Ru‐bpy[2].

   The field emission scanning electron microscope (FESEM) images
   exhibited in Figure [103]2b–d manifest the similar layered morphology
   of three COFs, which are hundreds of nanometers in size. The
   transmission electron microscope (TEM) image reveals that sp^2c‐COF,
   sp^2c‐COF‐Ir‐ppy[2], and sp^2c‐COF‐Ru‐bpy[2] possess a ribbon‐like
   layered structure, in good agreement with FESEM (Figure [104]2e–g).
   High‐resolution TEM (HRTEM) characterization of the same sample shows a
   cellular internal structure of COFs, with bright spots corresponding to
   the pores. The presence of evenly distributed Ir and Ru atoms in the
   framework was demonstrated using energy‐dispersive X‐ray spectroscopy
   in scanning transmission electron microscopy (TEM‐EDX)
   (Figure [105]2h–j). No Ir, Ru (or oxide) nanoparticles or clusters are
   detected in the spherical aberration‐corrected high‐angle annular
   dark‐field scanning TEM (SAC‐HAADF‐STEM) image. Single Ir and Ru atoms
   are identified in the SAC‐HAADF‐STEM image using Z‐contrast
   (Figure [106]2k–m).^[ [107]^32 ^]

   Furthermore, X‐ray absorption fine structure spectroscopy (XAFS) tests
   were conducted to certify the dispersion and coordination environment
   of single Ir and Ru atoms. Compared with IrO[2]/Ir foil and
   RuO[2]/Ru‐foil as references, the X‐ray absorption near‐edge structure