Selective magnetic resonance imaging of antibiotic-resistant bacteria leveraging ATP-binding cassette sugar transporter-responsive probes

Selective magnetic resonance imaging of antibiotic-resistant bacteria leveraging ATP-binding cassette sugar transporter-responsive probes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Selective magnetic resonance imaging of antibiotic-resistant bacteria leveraging ATP-binding cassette sugar transporter-responsive probes Yao He, Mengna Zhu, Yadan Zhao, Jiawei Zhang, Pengcheng Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5646556/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Current magnetic resonance imaging (MRI) methods often fail to differentiate bacterial infections from nonbacterial inflammatory conditions because of their poor specificity. To address this limitation, we synthesized two MRI probes that exploit bacterial-specific ATP-binding cassette (ABC) sugar transporters for the selective delivery of manganese porphyrin-based nanoparticles into antibiotic-resistant bacteria. These probes were synthesized via click chemistry by coupling azide-functionalized maltotriose with alkyne-modified manganese hematoporphyrin, which formed self-assembling nanoparticles. Our studies revealed ~ 65% probe uptake in gram-positive and gram-negative bacteria, with negligible uptake (~ 1%) in ABC transporter-deficient mutants. The probes demonstrated high longitudinal and transverse relaxivities (up to 11.56 mM⁻¹s⁻¹ and 102.0 mM⁻¹s⁻¹, respectively), enabling ultrasensitive MRI detection of human-derived methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli at concentrations as low as 10⁶ CFU. In murine models, the probes differentiated bacterial nephritis from nonbacterial inflammation and visualized bacteria within tumour tissues, outperforming clinically used gadolinium-based agents. This study provides a promising approach for precise magnetic resonance imaging of antibiotic-resistant bacterial infections in deep tissues. Biological sciences/Biological techniques/Imaging/Magnetic resonance imaging Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Physical sciences/Materials science/Biomaterials/Drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Antimicrobial resistance (AMR) represents a pressing global challenge, with biofilm-associated infections exacerbating diagnostic complexities and ranking among the leading causes of mortality, potentially resulting in 10 million deaths by 2050 1–4 . Current diagnostic approaches for bacterial infections rely primarily on blood tests and tissue biopsies, including bacterial culture, biochemical assays, immunoassays, polymerase chain reaction (PCR), and sequencing 5 – 9 . However, these techniques are often labor intensive and time consuming. Additionally, invasive biopsies for deep tissue infections introduce risks, such as sampling errors, which can compromise diagnostic accuracy. Noninvasive imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), offer rapid, real-time and deep-tissue visualization of infections without the need for invasive procedures 10 – 21 . Despite these advantages, these techniques exhibit limited specificity, making distinguishing bacterial infections from nonbacterial inflammatory conditions, such as cancer or autoimmune disorders, challenging. Recent advancements have introduced several targeted antimicrobial agents, including CRISPR‒Cas systems, engineered toxins, and stapled antimicrobial peptides 22 – 27 . Unlike these antimicrobial strategies, the “Trojan horse” antibiotic strategy—originally proposed in the 1980s—uses siderophore-linked antibiotics to exploit bacterial iron importers for intracellular delivery 28 – 36 . This nutrient-based antibiotic delivery mechanism significantly mitigates bacterial resistance 37 – 41 . However, since these strategies still rely on antibiotics, the risk of resistance development persists. An alternative approach leverages bacteria-specific ATP-binding cassette (ABC) sugar transporters to transport photosensitizers into bacterial cells by coupling them with unique carbon sources, such as maltose, maltotriose, or maltohexose 42 – 49 . Although these techniques enable both optical imaging and phototherapy for resistant infections, the limited tissue penetration of light (< 0.5 cm) restricts their application to superficial infections. A more promising but underexplored strategy involves delivering multifunctional MR probes selectively into bacterial cells to detect and treat antibiotic-resistant infections deep within tissues. To fill this gap, we synthesized a series of bacterial-targeted MR nanoprobes consisting of azide-modified maltotriose (MT) linked to alkyne-modified manganese haematoporphyrin (HP) and self-assembled to form nanoparticles (Fig. 1 a). As schematically illustrated in Fig. 1 b, bacteria internalize the synthesized MR probes by recognizing maltotriose as a carbon source. Through ABC sugar transporters—exemplified by Escherichia coli ( E. coli )—maltotriose uptake occurs via subunits such as LamB, MalE, MalF, MalG, and MalK. Specifically, LamB functions as an outer membrane porin, whereas MalE binds α (1–4)-glucosidically linked maltotriose for transport 50 – 53 . Analogously, our results confirm that various bacteria readily consume maltotriose-engineered MR probes disguised as nutrients. Upon internalization, manganese (Mn²⁺) within these probes produces MR signals by shortening T 1 and T 2 relaxation times, enhancing both bright and dark contrast in T 1 - and T 2 -weighted images, respectively. As the probes aggregate locally, they disrupt the magnetic field uniformity, accelerating transverse magnetization decay (M xy ) and generating a pronounced negative contrast effect 54 – 58 . In proof-of-concept studies, the developed MR nanoprobes enabled the detection of human-derived methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Escherichia coli (MDR E. coli ) at concentrations as low as ~ 10⁶ CFU. In addition to sensitive and specific imaging, haematoporphyrin in nanoprobes demonstrates ultrasound-activated antimicrobial activity, underscoring its potential to selectively target and treat bacterial infections in deep tissues. Results Design of bacteria-specific ABC sugar transporter-responsive MR nanoprobes. We synthesized two bacterial-specific ABC sugar transporter-responsive MR nanoprobes, i.e., MT-MnHPs and (MT) 2 -MnHPs, the detailed synthesis of which is described in the Supplementary Methods . Typically, MT-MnHP is a manganese-based haematoporphyrin compound featuring a tetramethylporphyrin core functionalized with a single maltoriose-modified triazole linker. (MT)₂-MnHP shares the tetramethylporphyrin core of MT-MnHP but includes a dual maltoriose-modified triazole linker. Briefly, an azide-functionalized maltotriose intermediate (compound 1 ) was synthesized at the anomeric carbon to facilitate subsequent functionalization via copper(I)-catalyzed click chemistry (Fig. 1 a). This intermediate was prepared following a modified protocol 48 , 59 ( Supplementary Figs. 1, 5 & 6 ). In parallel, alkyne-functionalized hematoporphyrins (compounds 2 and 3 ) were obtained via Williamson ether synthesis ( Supplementary Figs. 2, 7 & 8 ) and further chelated with manganese ions through a thermal solvent approach ( Supplementary Figs. 2, 9 & 10 ). These alkyne-derivatized manganese porphyrins were conjugated to the azide-functionalized maltotriose intermediate via click chemistry ( Supplementary Figs. 3 & 4 ). Through self-assembly, the final bacteria-targeting MR nanoprobes, MT-MnHPs and (MT) 2 -MnHPs, were successfully synthesized. Scanning transmission electron microscopy (STEM) images revealed that the synthesized nanoprobes exhibited a spherical morphology (Fig. 1 c). The hydrodynamic diameters of the MT-MnHPs and (MT) 2 -MnHPs, as measured by dynamic light scattering (DLS), were approximately 3.4 nm and 8.3 nm, respectively ( Supplementary Fig. 11 ). The slight discrepancy in particle size between DLS and TEM likely reflects differences in surface conditions under the two measurement techniques. Elemental mapping ( Supplementary Fig. 12 ) confirmed the uniform distribution of Mn, C, N, and O within the particles. While the manganese signals corresponded to the coordinated Mn ions, the carbon, nitrogen, and oxygen signals originated from the hematoporphyrin and maltotriose linkers. These results confirm the successful synthesis and nanoparticle formation of MT-MnHPs and (MT) 2 -MnHPs. To investigate the ability of MT-MnHPs and (MT) 2 -MnHPs to penetrate bacterial cells, MDR E. coli and MRSA were isolated from keratitis patients treated at the Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University. The isolated strains (~ 1.0 × 10⁸ CFU) were incubated with 0.3 mM solutions of MT-MnHPs or (MT) 2 -MnHPs at 37 ℃ for 2 hours, followed by several washes with phosphate-buffered saline (PBS). High-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) and elemental mapping revealed that while C, N, and O were detected in all bacterial samples, Mn signals were present exclusively in the groups treated with MT-MnHPs or (MT) 2 -MnHPs (Fig. 1 e). These results confirmed the internalization of the Trojan MR probes into bacterial cells. Ex vivo MRI scans of the treated bacterial samples were performed via a 3.0 T clinical MRI scanner with fast spin‒echo T 1 -weighted imaging (FSE T 1 WI) and T 2 -weighted imaging (FSE T 2 WI). A T 1 signal enhancement or T 2 signal decrease was observed exclusively in the bacteria treated with MT-MnHPs or (MT) 2 -MnHPs, with approximately twofold stronger signals than those in the untreated groups (Fig. 1 d, Supplementary Figs. 13 & 14 ). Notably, the MR images exhibited differential behavior: T 1 -weighted images displayed brighter signals, whereas T 2 -weighted images gradually darkened (Fig. 1 d). These findings demonstrate that MT-MnHPs and (MT) 2 -MnHPs function effectively as dual-mode MR probes and can successfully infiltrate bacterial cells, establishing their utility for T 1 /T 2 -weighted imaging applications. In vitro assessment of bacterial-specific ABC sugar transporter-responsive MR nanoprobes. The synthesized MR probes employed Mn(II) ions as the central components, which act as key MRI indicators. UV–vis absorption and fluorescence (FL) spectra were utilized to explore the interactions between Mn(II) ions and HP within both MT-MnHPs and (MT) 2 -MnHPs. The absorption spectrum of HP (Fig. 2 a) displayed a characteristic Soret band (393 nm) and four Q-bands (496, 531, 566, and 619 nm), corresponding to electron transitions from the HOMO to the π* orbitals. While both probes retained these typical bands, MT-MnHPs and (MT) 2 -MnHPs also exhibited Soret peaks at 368 or 371 nm, along with two Q-bands spanning 540–600 nm, and introduced a novel peak at 460 nm. The emergence of these two Q-bands confirmed the successful coordination of manganese ions to the porphyrin core, where two Q-bands resulted from π-to-π* transitions within the aromatic porphyrin framework. Under 370 nm light excitation, both probes presented two weak fluorescence peaks (Fig. 2 b), with lower intensities than those of pure HP, indicating energy transfer from HP to Mn ions and maltotriose. A higher maltotriose content increased energy transfer, confirming that Mn(II) ions selectively coordinate with the porphyrin ring and that MT-MnHPs exhibited superior optical activity relative to that of (MT) 2 -MnHPs. The impact of maltose linkages on sonodynamic therapy (SDT) efficiency was investigated by measuring singlet oxygen (¹O₂) production via SOSG. MT-MnHPs generated ¹O₂ under ultrasound (US) irradiation, with the yield increasing over time and reaching a plateau after 10 minutes (Fig. 2 c). This plateau established 10 minutes as the optimal excitation period. Notably, MT-MnHPs outperformed (MT) 2 -MnHPs in generating ¹O₂ ( Supplementary Fig. 15 ), suggesting that differences in maltose linkages may influence ROS generation efficiency. The sonotoxicity of MT-MnHPs against MRSA was assessed via a turbidity assay following 10 minutes of US exposure. As shown in Supplementary Fig. 16 , bacterial inhibition correlated with increasing concentrations of HP, MT-MnHPs and (MT) 2 -MnHPs. At 0.3 mM, MT-MnHPs completely inhibited bacterial growth, whereas HP and (MT) 2 -MnHPs achieved only 29% and 67% inhibition, respectively. Although both probes produced ROS under US, HP failed to penetrate bacteria, and (MT) 2 -MnHPs demonstrated weaker ROS production, limiting their antibacterial efficacy. Motivated by the successful coordination of Mn ions to the porphyrin core, we next examined the MRI contrast properties. As the concentration of either MT-MnHPs or (MT) 2 -MnHPs increased from 0 to 0.3 mM, the T 1 -weighted MR images brightened, whereas the T 2 -weighted images dimmed (Fig. 2 d). This dual contrast behavior was corroborated by corresponding signal intensity changes, with increasing T 1 signals and decreasing T 2 signals ( Supplementary Fig. 17 ). In addition, MT-MnHPs showed a marked MRI contrast with a relaxivity r 1 as high as 11.56 mM − 1 s − 1 ( Supplementary Fig. 18a ), which is almost 3.0-fold greater than that of (MT) 2 -MnHPs. In addition, the r 2 value of MT-MnHPs is 102.0 mM − 1 s − 1 ( Supplementary Fig. 18b ), which is significantly greater than that of (MT) 2 -MnHPs. Additionally, changes in the signal-to-noise ratios (ΔSNRs) were obtained by quantifying the differences in signal enhancements in the images in Fig. 2 d for a better comparison of the MRI performance of the NPs. The ΔSNR of MT-MnHPs was much higher than that of (MT) 2 -MnHPs at all concentrations above 0.075 mM ( Supplementary Fig. 18c ), which further indicates the significantly enhanced MRI performance of MT-MnHPs. Proton–proton or proton–electron interactions involving water protons and paramagnetic ions or molecules predominantly govern the functionality of paramagnetic centres as contrast agents 60 , 61 . To elucidate the molecular-level MRI contrast characteristics of MT-MnHPs and (MT) 2 -MnHPs, density functional theory (DFT) calculations were conducted via the b3lyp/def2SVP functional 62 . Additionally, the independent gradient model based on Hirshfeld partitioning (IGMH) was applied through the Multiwfn program 63 – 66 . The analysis revealed the presence of intermolecular forces, including attractive, repulsive, and van der Waals interactions, between water molecules and MT ligands in MT-MnHPs or (MT)₂-MnHPs, as depicted in Fig. 2 e (I and II). Both MT-MnHPs and (MT)₂-MnHPs exhibited weak noncovalent interactions (green arrows in Fig. 2 e), which facilitated the enrichment of bound water molecules near the paramagnetic centers. This binding restricts the movement of water molecules, thereby mitigating the averaging effect and shortening their relaxation times. Furthermore, the binding energy between water and MT ligands in MT-MnHPs (79.8 kJ/mol) exceeds that in (MT)₂-MnHPs (73.2 kJ/mol), underscoring the superior MRI contrast performance of MT-MnHPs. Most conventional treatments fail to eliminate biofilms because of limited bactericidal penetration. Given the demonstrated bacterial targeting ability and superior sonodynamic activity of MT-MnHPs, we investigated their potential to disrupt biofilms. MRSA biofilms served as the model for this proof-of-concept study. Using FITC-labelled biofilms, we observed that MT-MnHPs combined with US generated red fluorescence at the depth of penetration after 2 minutes of US exposure, indicating efficient penetration driven by bacterial uptake and ultrasound-assisted diffusion (Fig. 2 f). Crystal violet staining was used to quantify the sonodynamic effect on biofilm integrity. As illustrated in Fig. 2 g, untreated biofilms remained thick and structurally intact, whereas those treated with MT-MnHPs and subjected to increasing US irradiation times presented significant reductions in thickness and cohesion. Quantitative analysis (Figs. 2 h– 2 j and Supplementary Fig. 19 ) revealed that MT-MnHPs + US eradicated 97% of the MRSA biofilms, outperforming (MT) 2 -MnHPs + US, which cleared 78% of the MRSA biofilms. Similarly, compared with (MT) 2 -MnHPs, MT-MnHPs achieved 97% clearance of MDR E. coli biofilms, whereas (MT) 2 -MnHPs achieved 80% clearance. These findings establish MT-MnHPs as effective probes for sonodynamic biofilm eradication. Bacterial-specific ABC sugar transporter-responsive MR nanoprobes target diverse bacterial species. To optimize the incubation time, we systematically investigated the uptake efficiency of MT-MnHPs (0.3 mM) by bacteria through flow cytometry. As shown in Supplementary Fig. 20 , the uptake of ~ 1.0 × 10⁸ CFU of MRSA and MDR E. coli reached maximum levels—73.7% for MRSA and 62.4% for MDR E. coli —after 2 hours of incubation. Extending the incubation period yielded no significant increase in uptake, indicating that saturation had been achieved. Therefore, all following experiments were performed after incubating with MT-MnHPs (0.3 mM) for 2 h. Next, we assessed the ability of MT-MnHPs to target a range of natural bacterial strains. We selected two gram-negative species—MDR E. coli and Salmonella typhimurium (STm)—and two gram-positive species—MRSA and Staphylococcus aureus ( S. aureus ). Confocal laser scanning microscopy (CLSM) images (Fig. 3 a and Supplementary Fig. 21 ) revealed prominent red fluorescence signals from MT-MnHPs (emission: 600–700 nm, excitation: 370 nm) in all four bacterial strains after incubation with MT-MnHPs (0.3 mM) for 2 hours. Quantitative analysis via flow cytometry confirmed uptake efficiencies of 73.7% for MRSA, 62.4% for MDR E. coli , 64.9% for S. aureus , and 72.5% for STm. In contrast, negligible fluorescence was observed when bacteria were treated with unmodified HP ( Supplementary Fig. 22 ), confirming that maltotriose modification is essential for bacterial targeting. We further explored whether MT-MnHPs rely on ABC sugar transporters for bacterial uptake. Two bacterial mutants—ΔlamB and ΔmalE—were generated and validated through Sanger sequencing ( Supplementary Notes ). As anticipated, no fluorescence signals were detected in the ΔlamB or ΔmalE mutants treated with MT-MnHPs (Fig. 3 b), which is consistent with the CLSM observations. Additionally, competition assays demonstrated that preincubation of bacteria with increasing concentrations of maltotriose (0, 0.3, or 0.6 mM) for 5 min significantly diminished MT-MnHP uptake (Fig. 3 c). These findings confirm that MT-MnHPs enter bacteria through the ABC sugar transporter pathway. We next assessed the specificity of MT-MnHPs for bacteria over mammalian cells. HeLa cells and human blood samples spiked with MRSA were treated with MT-MnHPs (0.3 mM) for 2 hours and washed with PBS. As shown in Supplementary Fig. 23 , red fluorescence was detected only in bacterial cells, with no signal observed in HeLa cells or blood cells, indicating minimal uptake by mammalian cells. This selectivity arises from the absence of ABC sugar transporters in mammalian cells. To determine the detection limit, we imaged serial dilutions of MRSA and MDR E. coli incubated with the MR probes (Figs. 3 d– 3 g). Bacterial concentrations as low as ~ 10⁶ CFU produced detectable signals ( P < 0.001), highlighting the sensitivity of the approach. These results demonstrate the potential of bacteria-targeting MR nanoprobes for clinical bacterial diagnostics. Magnetic resonance imaging of bacterial nephritis in mice via bacterial-specific ABC sugar transporter-responsive MR nanoprobes. We evaluated the feasibility of the developed MR nanoprobes for imaging bacteria residing deep within tissues. To establish a proof-of-concept model, we induced nephritis in mice via MRSA infection. As shown schematically in Fig. 4 a, female mice (6–8 weeks old, n = 3) were injected with 25 µL of MRSA, followed 12 hours later by intravenous administration of 200 µL of PBS, HP (0.3 mM), MT-MnHPs (0.3 mM), or (MT) 2 -MnHPs (0.3 mM). The concentration of MRSA at the infection site, measured by harvesting and culturing kidney tissue, was ~ 1.0 × 10 8 CFU. We observed that the strongest MR signal was localized exclusively at the infected site in the mice treated with MT-MnHPs (Fig. 4 b). Quantitative analysis revealed that the T 1 or T 2 signal intensities in the MT-MnHP-treated group were approximately twice those observed in the (MT) 2 -MnHP-treated group (Fig. 4 c). Notably, the developed MR nanoprobes demonstrated the ability to detect bacterial concentrations as low as ~ 1.0 × 10⁶ CFU within the kidney ( P < 0.001), as shown in Supplementary Fig. 24 —sensitivity adequate for many in vivo scenarios. To determine the specificity of the developed MR nanoprobes for bacterial nephritis, we developed a comparative model of glycerin-induced nephritis. As depicted in Fig. 4 d, female mice (6–8 weeks old, n = 3) were injected in situ with 25 µL of 50% (v/v) glycerin, followed by an intravenous injection of 200 µL of MT-MnHPs (0.3 mM) 12 hours later. As expected, T 1 and T 2 signal changes were significantly greater in the mice with MRSA nephritis than in those with glycerin-induced nephritis or in the control mice treated with MT-MnHPs ( P < 0.001) (Figs. 4 e & 4 f). Furthermore, we compared the imaging performance of the developed MR nanoprobes with that of gadopentetic acid (Gd-DTPA), a clinically used contrast agent. Both MRSA-infected and glycerin-nephritis-bearing mice were scanned with a 3.0 T MRI scanner via a T 1 -weighted sequence (Figs. 4 g & 4 h). Consistent with prior reports 67 , 68 , Gd-DTPA exhibited strong T 1 signal enhancement due to the presence of Gd³⁺, which promoted longitudinal proton relaxation through unpaired electrons and extended the electron spin relaxation time. However, unlike the bacterial-specific ABC sugar transporter-responsive MR nanoprobes, which selectively enhanced signals only at sites of bacterial nephritis, Gd-DTPA increased T 1 signals in both the bacterial and nonbacterial nephritis models (Fig. 4 g). These results underscore the high selectivity of the developed MR nanoprobes, which can effectively distinguish bacterial nephritis from other inflammatory conditions. The specificity and tunable sensitivity of the developed method are attributed to the preferential internalization of the developed MR nanoprobes within bacterial cells. Magnetic resonance imaging of bacteria in tumours via bacterial-specific ABC sugar transporter-responsive MR nanoprobes. Next, we verified the effectiveness of the developed MR nanoprobes for imaging bacteria in tumours. Accordingly, we constructed proof-of-concept models of bacteria in tumour xenografts. To construct the tumour xenograft model, we subcutaneously injected 100 µL of CT26 cells (~ 5 × 10 6 cells) into the right back region of female nude mice (6–8 weeks old). When the tumours grew to 100 mm 3 , we subcutaneously injected 50 µL of MRSA into the left thigh region of the mice or into both the left thigh region and the right tumour region of the mice (Figs. 5 a & 5 b), followed by intravenous injection of 200 µL of PBS or MT-MnHPs (0.3 mM). The infected sites as well as the tumour sites were then imaged by a 3.0 T MRI scanner using a T 1 -weighted sequence and T 1 -weighted sequence at 6 h postinjection of MT-MnHPs (Figs. 5 c & 5 d). As revealed in Figs. 5 c & 5 d, we observed MR signals only at the infected sites instead of at the tumour sites containing no bacteria, indicating that the developed MR nanoprobes enabled the discrimination of bacteria from tumors. As expected, the detection signals from the infected sites treated with MT-MnHPs were significantly stronger than those from their counterparts treated with PBS were (e.g., ~ 2.0 (MRSA, left)-fold increase in T 1 -weighted signals and ~ 2.1 (MRSA, left)-fold decrease in T 2 -weighted signals) (Figs. 5 c & 5 e). As further revealed in Figs. 5 d & 5 f, we observed MR signals simultaneously at the infected sites and the tumor sites containing bacteria. Consistently, the signals detected from both the infected sites and the tumour sites containing bacteria treated with MT-MnHPs were much stronger than those detected with PBS (e.g., ~ 2.7 (MRSA, left) and ~ 2.1 (tumor + MRSA, right)-fold increase in T 1 -weighted signals and ~ 1.8 (MRSA, left) and ~ 1.8 (tumor + MRSA, right)-fold decrease in T 2 -weighted signals). These results together prove that the developed MR nanoprobes allowed the imaging of bacteria residing within tumour tissues. In vitro antibacterial activity of the developed strategy. In addition to enabling MRI of bacterial infections, the synthesized MT-MnHPs (0.3 mM) exhibited potent antibacterial effects due to their sonodynamic properties. We verified this antimicrobial activity through a live/dead bacterial staining assay. In this assay, the green fluorescent dye NO 1 (λ ex = 488 nm, λ em = 525 nm) marked both intact (viable) and damaged (nonviable) bacterial membranes, whereas propidium iodide (PI, λ ex = 536 nm, λ em = 617 nm) selectively penetrated compromised membranes, emitting red fluorescence to indicate bacterial cell death. As demonstrated in Supplementary Fig. 25 , green fluorescence dominated in the control groups, whereas treatment with MT-MnHPs or (MT) 2 -MnHPs under SDT elicited increased red fluorescence, confirming bacterial membrane disruption. Scanning electron microscopy (SEM) images provided further morphological evidence of bacterial damage ( Supplementary Fig. 26 ). MRSA and MDR E. coli cells incubated with MT-MnHPs (0.3 mM) for 2 hours, followed by ultrasound irradiation (1 MHz, 1.5 W/cm²) for 10 minutes, displayed wrinkling or lysis of the cell membranes. These effects were more pronounced with MT-MnHPs than with (MT) 2 -MnHPs, aligning with their superior singlet oxygen ( 1 O₂) generation, which enhanced the SDT efficacy. We next investigated the penetration depth of ultrasound in modulating the antibacterial efficiency of MT-MnHPs. Using chicken breast tissues of varying thicknesses (0 to 4.0 cm) placed over bacterial cultures in 24-well plates ( Supplementary Fig. 27b ), we performed agar plate experiments following ultrasound irradiation (1 MHz, 1.5 W/cm²) for 10 minutes. As shown in Supplementary Figs. 27a-27c , the antibacterial efficacy decreased as the tissue thickness increased. When the tissue was 4.0 cm thick, the antibacterial rates were approximately 65% for MRSA and 61% for MDR E. coli . In contrast, the PBS-treated groups showed no antibacterial effects with increasing tissue thickness. These results highlight the robust penetration depth of MT-MnHPs, establishing a strong basis for future applications of SDT in deep tissues. The superior sonodynamic performance of MT-MnHPs compared with that of (MT) 2 -MnHPs underscores their potential for in vivo therapeutic interventions. In vivo antibacterial activity of the developed strategy. To assess the in vivo antibacterial efficacy of the developed strategy, we evaluated its performance in a proof-of-concept bacterial nephritis model in mice. As illustrated schematically in Fig. 6 a, MRSA (1.0 × 10⁸ CFU) was injected intravenously into female mice (6–8 weeks old, n = 3). At 12 hours post infection, the mice received 200 µL of MT-MnHPs (0.3 mM) or PBS through intravenous injection on days 1, 3, 5, and 7. Four hours after each injection, we applied US to initiate SDT, with each session lasting 10 minutes. Imaging was performed following each irradiation to monitor therapeutic progress in real time. Posttreatment, infected kidney tissues were excised, homogenized, and cultured on agar plates to quantify the bacterial load. As shown in Figs. 6 b & 6 c, only a few colonies remained in the MT-MnHPs + US group after seven days of treatment. The antibacterial rate of MT-MnHPs under ultrasound reached 96% against MRSA. Consistent with these findings, hematoxylin‒eosin (H&E) staining (Fig. 6 d) revealed intact tissue architecture and the absence of cell necrosis exclusively in the MT-MnHPs + US group. This outcome highlights the therapeutic benefit of SDT in achieving high antibacterial efficacy. Additionally, the developed strategy enabled real-time visualization of the antibacterial effects. MRI was performed after each irradiation session to assess therapeutic outcomes dynamically. As depicted in Figs. 6 e & 6 f, the MR signal gradually diminished as the treatment progressed, which was correlated with reductions in the bacterial load ( P < 0.001). Quantitative analysis confirmed that the decrease in MR signal intensity was directly proportional to bacterial clearance. This alignment between MR signal changes and bacterial counts underscores the utility of bacterial concentration-dependent MRI as a reliable marker for monitoring both detection and treatment efficacy. Together, these data validate the adaptive antibacterial potential of this strategy in vivo, demonstrating its feasibility for precise and effective therapeutic applications. Toxicity assessment of bacterial-specific ABC sugar transporter-responsive MR nanoprobes. We comprehensively evaluated the toxicity profiles of MT-MnHPs and (MT) 2 -MnHPs. As shown in Supplementary Fig. 28a , methyl thiazolyl tetrazolium (MTT) assays revealed that the viability of CT26, mREC, HeLa, and MCF-7 cells exceeded 80% following incubation with either MT-MnHPs (0.3 mM) or (MT) 2 -MnHPs (0.3 mM). These findings indicate that both probes exhibit minimal cytotoxicity under the tested conditions. We further assessed hemolytic activity by exposing red blood cells to MT-MnHPs and (MT) 2 -MnHPs at concentrations up to 0.3 mM. As shown in Supplementary Fig. 28b , the hemolysis rates were 9.8% for MT-MnHPs and 10.8% for (MT) 2 -MnHPs, indicating favourable hemocompatibility. Additionally, H&E staining of major organs ( Supplementary Fig. 28c ) harvested 24 hours postinjection revealed no histopathological abnormalities, further confirming the negligible in vivo toxicity of both probes. To assess systemic toxicity, we conducted blood biochemistry and routine hematological analyses at the administered dose. As shown in Supplementary Figs. 29a-29h , all biochemical parameters for the MT-MnHP and (MT) 2 -MnHP groups remained within normal ranges and were comparable to those of the PBS control group. Ex vivo fluorescence imaging ( Supplementary Fig. 30a ) further revealed that the probes predominantly accumulated in the liver and kidneys at 0.5 hours postinjection, with negligible fluorescence signals detected in other organs. Importantly, the fluorescence signals had almost entirely disappeared by 48 hours, suggesting efficient clearance. Furthermore, strong fluorescence signals were detected in urine samples collected 24 hours postinjection ( Supplementary Fig. 30b ), confirming renal excretion as a primary elimination route. These results collectively demonstrate that MT-MnHPs and (MT) 2 -MnHPs exhibit minimal toxicity, high biocompatibility, and effective clearance, supporting their potential for in vivo applications. Discussion The ABC sugar transporter-responsive MR nanoprobes presented here demonstrate a potent and selective approach for diagnosing and treating human-derived antibiotic-resistant bacterial infections in deep tissues. By harnessing maltotriose-coupled manganese porphyrin derivatives, we enabled selective bacterial targeting through ABC sugar transporters, facilitating robust MR contrast and ultrasound-activated antimicrobial activity. This bacterial-specific uptake mechanism enables visualization of bacterial distribution within deep tissues and has potential for combined diagnostic and therapeutic applications. Our findings address the long-standing limitation of the poor specificity of conventional MRI for bacterial infections. Despite these promising outcomes, several limitations warrant consideration. First, while in vitro and murine models have validated the selective uptake and therapeutic efficacy of these MR probes, further studies are needed to verify their effectiveness and safety in larger animal models and human tissues. The bacterial strains tested, although representative, do not cover the full spectrum of clinically relevant pathogens, and variation in ABC sugar transporter expression across bacterial species may influence probe uptake. Additionally, while ultrasound-activated antimicrobial effects have potential, the depth of ultrasound penetration may limit its therapeutic efficacy in larger or denser tissues. To optimize this strategy for clinical applications, future studies should evaluate the safety profile and pharmacokinetics of these agents across a range of bacterial infections and tissue types. Enhancing probe specificity and retention within target tissues, refining ultrasound parameters to maximize tissue penetration, and developing real-time imaging protocols could further strengthen the translational potential of this approach. Methods Preparation and purification of bacterial-specific ABC sugar transporter-responsive MR nanoprobes. Refer to Fig. 1 . MT-MnHPs and (MT)₂-MnHPs were prepared by coupling alkyne-functionalized manganese porphyrins (compound 2 ) with azide-functionalized maltotriose (compound 1 ) through a copper-catalyzed azide‒alkyne cycloaddition (click reaction). The detailed synthesis protocols and characterizations of intermediates 1 , 2 , and 3 are provided in the Supplementary Information. The specific click reaction between 1 and 2 to generate MT-MnHPs is described as follows: Compound 1 (50 mg, 0.05 mmol) and compound 2 (69 mg, 0.10 mmol) were dissolved in DMF (10 mL). CuI (0.2 mg, 1.0 µmol) and DIPEA (1.2 mg, 0.01 mmol) were then added to the solution. The reaction mixture was stirred at ambient temperature under nitrogen for 12 hours. The solvent was removed under reduced pressure, and the residue was dissolved in CH₂Cl₂ (30 mL). The organic phase was washed sequentially with water (10 mL) and saltwater (10 mL), dried over Na₂SO₄, filtered, and evaporated to dryness under vacuum. The resulting acetyl-protected intermediate was deprotected in a mixture of CH₃OH (2 mL) and aqueous LiOH (1.0 M, 2 mL) under nitrogen for 24 hours. The crude (MT)₂-MnHP were neutralized via Dowex 50 W resin, filtered, and concentrated under vacuum. MT-MnHP was further purified via silica gel flash chromatography. To obtain MT-MnHP nanoparticles, 10 mg of MT-MnHP was dissolved in 1 mL of DMSO and added dropwise to 9 mL of deionized water under sonication with magnetic stirring. The suspension was stirred overnight, followed by centrifugation (10,000 rpm, 10 min) and washing with deionized water. A detailed characterization of MT-MnHPs and the synthesis and characterization of (MT)₂-MnHPs can be found in the Supplementary Information. Characterizations of bacterial-specific ABC sugar transporter-responsive MR nanoprobes. We evaluated the morphology and size of bacteria-targeting MR probes via transmission electron microscopy (TEM) at 200 kV (Philips CM 200). UV‒visible (UV‒vis) absorption spectra were captured on a Perkin-Elmer Lambda 750 UV‒vis/NIR spectrophotometer. Photoluminescence spectra were recorded via a HORIBA Fluoromax-4 spectrofluorometer. Dynamic light scattering (DLS) measurements were taken with a Delsa™ Nano submicron particle analyser. For bacterial fluorescence imaging in vitro, we used a Leica TCS-SP5 II confocal laser scanning microscope (CLSM). Ex vivo and in vivo MR images were acquired with a 3.0 T clinical MRI scanner utilizing fast spin echo (FSE) T 1 -weighted (T 1 WI) and T 2 -weighted (T 2 WI) sequences. Bacterial culture. Multidrug-resistant (MDR) E. coli and methicillin-resistant Staphylococcus aureus (MRSA), which were isolated from keratitis patients, were obtained from the Eye, Ear, Nose and Throat Hospital, Fudan University, with ethical approval (EENTIRB-2017-06-07-01). Staphylococcus aureus and Salmonella typhimurium (BNCC 108207) were sourced from the American Type Culture Collection and BeNa Culture Collection, respectively. Bacteria were cultured in Luria–Bertani (LB) medium, initially rehydrated in liquid LB, plated on LB agar, and incubated at 37 ℃ for 12 hours. A single colony was picked and cultured in LB medium at 250 rpm and 37 ℃. Bacterial cells at exponential growth were collected, and concentrations were confirmed by optical density at 600 nm. Colony numbers were quantified with a colony counter (Czone 8). The protocols conformed to the Declaration of Helsinki and Chinese regulations. Minimum inhibitory concentration (MIC) of MT-MnHPs MDR E. coli and MRSA were cultured in LB media at 37 ℃ on a shaker (200 rpm) for 4–6 hours. The bacterial concentrations were adjusted to an OD 600 of 0.1 for 1 × 10⁸ CFU/mL. Bacterial suspensions (20 µL of 1 × 10⁹ CFU/mL) were added to PBS (160 µL per well). Phosphate-buffered saline (PBS), HP, MT-MnHPs, or (MT) 2 -MnHPs were added to a Corning 96-well plate and incubated at 37 ℃ and 200 rpm for 18 hours. OD 600 readings were recorded with a Tecan Infinite M200 microplate reader, with triplicate cultures for each assay, which was repeated at least twice. In vitro cellular experiments. A mouse colorectal cancer cell line (CT26 cells) and human cervical cells (HeLa cells) were cultured in 1640 medium. Human breast cancer cells (MCF-7 cells) and mouse retinal endothelial cells (mRECs), cultured in Dulbecco’s modified Eagle’s medium with high glucose (H-DMEM), were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (China). All the abovementioned media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% relevant antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin). All the cell lines were cultured at 37 ℃ in a 5% CO 2 incubator with a humidified atmosphere. Human studies Blood samples were obtained from a healthy donor who provided written informed consent. All procedures involving human blood were conducted with approval from Soochow University's ethics committee in compliance with institutional and legal standards. Vitreous fluid samples (0.1 mL, nondiluted and sterile) were drawn from ten patients diagnosed with bacterial endophthalmitis during diagnostic pars plana vitrectomy (PPV) procedures. These samples were collected via a 30 G needle and transferred immediately to presterilized microfuge tubes for imaging. Additional clinical samples were provided by the Eye Bank at the Eye, Ear, Nose, and Throat Hospital, Fudan University, with ethics committee approval (EENTIRB-2017-06-07-01). CLSM 3D imaging and crystal violet staining of biofilms. MRSA, diluted to 10⁵ CFU/mL (OD 600 = 0.025), was added (120 µL) to 96-well plates and cultured at 37 ℃ for 24 hours. After washing with PBS, mature biofilms were treated with PBS or MT-MnHPs (0.3 mM), followed by ultrasound exposure (1 MHz, 1.5 W/cm², 0–2 min). The biofilms were fixed in 4% glutaraldehyde for 4 hours, stained with ethidium bromide (EB) and FITC-ConA at 4 ℃ for 15 minutes, and observed via CLSM 3D imaging. Mature biofilms were prepared as described above. A 2 mL aliquot of bacterial suspension was added to a 12-well growth plate containing sterile coverslips positioned vertically and incubated under static conditions at 37 ℃ for 24 hours. Afterward, the biofilms were rinsed multiple times with sterile PBS and incubated with 2 mL of PBS, MT-MnHPs (0.3 mM), or (MT) 2 -MnHPs (0.3 mM) for 2 hours at 37 ℃. The biofilms were then subjected to US exposure (1 MHz, 1.5 W cm⁻²) for 10 minutes. Following solvent removal, 2 mL of absolute methanol was applied to fix the biofilm for 15 minutes, and the sample was subsequently stained with 2 mL of crystal violet (0.5%, v/v) for an additional 15 minutes. The residual biofilm was then rinsed with PBS and completely dissolved in 2 mL of an acetic acid solution (33%, v/v) for 10 minutes. The absorbance of the resulting solution was measured at 590 nm with a microplate reader. The residual biofilm percentage was quantified as the ratio of the absorbance of the material- or US-treated samples relative to the control absorbance. The experiments were conducted in triplicate to minimize experimental error. In vitro imaging of bacteria. A purified, resuspended bacterial suspension (20 µL, 1.0 × 10⁷ CFU) was incubated with MT-MnHPs (0.3 mM, 200 µL), (MT) 2 -MnHPs (0.3 mM, 200 µL), HP (0.3 mM, 200 µL), maltotriose (0.3 mM, 200 µL), or PBS (200 µL) for 2 hours in a shaking incubator at 200 rpm and 37 ℃. Bacteria were then collected by centrifugation at 6000 rpm for 5 minutes in Eppendorf (EP) tubes, resuspended, and washed three times with PBS. A 10 µL sample of the washed bacterial suspension was then transferred onto a microscope slide, covered with a coverslip, and imaged via confocal laser scanning microscopy (CLSM, Leica, TCSSP5 II) with a diode laser at 30% power. All fluorescence images were captured with CLSM under identical optical parameters, with consistent brightness and contrast applied automatically. Region-of-interest (ROI) processing and analysis were conducted via Leica Application Suite Advanced Fluorescence Lite software. In vivo imaging of bacteria. All procedures involving animals were approved by the Soochow University Laboratory Animal Center (SYXK(SU) 2021–0073). To establish MRSA-induced nephritis, 25 µL of MRSA was directly injected into the kidneys of nude mice (female, 6–8 weeks old; n = 3). Twelve hours postinjection, infected mice received intravenous administration of MT-MnHPs (0.3 mM, 200 µL), (MT) 2 -MnHPs (0.3 mM, 200 µL), HP (0.3 mM, 200 µL), or PBS (200 µL). In vivo MRI was then performed 30 minutes after injection via a 3.0 T clinical MRI scanner, employing FSE T 1 -weighted imaging (TE = 12 ms; TR = 400 ms; FOV = 80 × 80 mm; slice thickness = 2 mm; spacing = 0.2 mm; matrix = 256 × 256) and T 2 -weighted imaging (TE = 58 ms; TR = 300 ms; FOV = 80 × 48 mm; slice thickness = 2 mm; spacing = 0.2 mm; matrix = 256 × 256). The local MRSA concentration at the infection site during imaging was approximately 1.0 × 10⁸ CFU, as confirmed through kidney tissue homogenization and CFU quantification. Additionally, to assess selectivity, glycerin-induced nephritis was modelled in mice (female, 6–8 weeks old, n = 3) and treated with either MT-MnHPs (0.3 mM, 200 µL) or Gd-DTPA (0.2 mL kg⁻¹, 200 µL) for MRI (T 1 -weighted: TE = 12 ms; TR = 400 ms; FOV = 80 × 80 mm; slice thickness = 2 mm; spacing = 0.2 mm; matrix = 256 × 256). To construct a tumour-containing bacterial model, we subcutaneously injected 100 µL of CT26 cell suspension into the right back region of nude mice (female, 6–8 weeks old, n = 3). When the tumor size reached ~ 100 mm 3 , the mice were randomly divided into two groups. In one group, we subcutaneously injected bacteria into the left thigh (MRSA: 50 µL, ~ 1.1 × 10 7 CFU) but not into the right tumour. In the other group, we subcutaneously injected bacteria into the left thigh (MRSA: 50 µL, ~ 1.1 × 10 7 CFU) as well as the right tumour (MRSA: 50 µL, ~ 1.1 × 10 7 CFU). The actual number of bacteria at the infection sites during imaging was also determined via tissue harvesting, homogenization and culture with a CFU count. The final concentration of MRSA at the infection site during imaging was ~ 1.0 × 10 7 CFU. Twelve hours postinjection, infected mice received intravenous administration of MT-MnHPs (0.3 mM, 200 µL). In vivo MRI was then performed 4 hours after injection via a 3.0 T clinical MRI scanner, employing FSE T 1 -weighted imaging (TE = 17.2 ms; TR = 629 ms; FOV = 3 × 3 cm 2 ; slice thickness = 1.5 mm; spacing = 0.2 mm; matrix = 256 × 256) and T 2 -weighted imaging (TE = 42.1 ms; TR = 2500 ms; FOV = 3 × 3 cm 2 ; slice thickness = 1.5 mm; spacing = 0.2 mm; matrix = 256 × 256). In vitro antibacterial assays. The bacterial suspensions were treated under identical conditions, followed by staining with 15 µL of N01/PI solution (1 mg mL⁻¹) for 15 minutes in darkness. The stained bacteria were rinsed with sterile PBS to remove residual dye and pelleted by centrifugation (6000 rpm, 5 minutes). Viability and cell death were then visualized via CLSM. Bacterial samples treated with PBS or ultrasound alone served as controls. SEM was used to characterize the morphology of bacteria treated with PBS, (MT) 2 -MnHPs (200 µL, 0.3 mM), or MT-MnHPs (200 µL, 0.3 mM) after ultrasound exposure (1 MHz, 1.5 W cm⁻², 10 minutes). The treated bacterial suspensions were placed on silicon wafers, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and subsequently dehydrated via a graded ethanol series (50%, 75%, 90%, and 100%, each for 5 minutes). The silicon wafers were dried thoroughly and coated with gold prior to SEM imaging. In a 24-well plate setup, chicken breast tissues of varying thicknesses were covered with MRSA or MDR E. coli incubated with MT-MnHPs (0.3 mM). Following ultrasound treatment (1 MHz, 1.5 W cm⁻² for 10 minutes), agar plate assays were conducted to quantify bacterial viability. The antibacterial rate was calculated on the basis of CFU counts on agar plates as follows: Antibacterial rate (%) = ( N control - N experiment )/ N control ×100% (1) where “ N control ” and “ N experiment ” represent bacterial counts (CFU mL − 1 ) in the control groups of “PBS” and other experimental groups (experiment), respectively. In vivo antibacterial assays. We used MRSA-induced nephritis in mice to evaluate the antibacterial ability of the developed strategy in vivo. To construct the model, MRSA (25 µL, ~ 1.1 × 10 8 CFU) was injected in situ into the right kidney of each mouse (female, 6–8 weeks old, n = 3). At 12 h postinjection, these mice were intravenously injected with 200 µL of 0.3 mM MT-MnHPs or PBS buffer on days 1, 3, 5 and 7. Six hours after each drug injection, the infected sites were irradiated with or without ultrasound (1 MHz, 1.5 Wcm − 2 , 10 min). After SDT, the infected kidney tissues were extracted, homogenized, and cultured on plates. The corresponding antibacterial rate was calculated via Eq. (1). After treatment, the infected kidney tissues were fixed in 4% PFA solution for H&E staining. Statistical analysis. For statistical significance testing, we used one-way ANOVA or the paired two-tailed t test. The statistical analysis was performed via Origin or GraphPad Prism software. The error bars represent the standard deviation obtained from three independent measurements. All imaging experiments were repeated three times with similar results. A region of interest (ROI) was employed for quantitative assessments of fluorescence intensity, which was calculated via commercial image analysis software (Leica Application Suite Advanced Fluorescence Lite, LAS AF Lite). Life Science Reporting Summary. Further information on experimental design is available in the Life Science Reporting Summary. Declarations Acknowledgements We thank Prof. Ling Wen (Soochow University, China) for her general help and valuable suggestions. Y. H. discloses support for the research described in this study from the National Key R&D Program of China (2023YFB3208200), the National Natural Science Foundation of China [grant numbers 22393932, T2321005, 21825402], the Science and Technology Development Fund, Macau SAR [grant number 0002/2022/AKP, 0115/2023/RIA2], the Major Independent Research Project of Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices (grant number L421490022) and the Program for Jiangsu Specially Ap-pointed Professors to Professor Yao He, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), 111 Project and Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO-CIC). H.Y.W. provided support for the research described in this study from the National Natural Science Foundation of China [grant number 22074101]. Author Contributions M.N.Z., Y.D.Z., J.W.Z., B.S., H.Y.W. and Y.H. conceived and designed the research. M.N.Z., Y.D.Z. and J.W.Z. carried out most of the experiments and analysed the data. P.C.W., J.L.Z., Y.Y.Z. and X.L. performed additional experiments and characterizations. M.N.Z., J.W.Z., Y.D.Z., B.S., H.Y.W. and Y.H. wrote the manuscript. Competing interests The authors declare no competing financial interests. Additional information Supplementary Information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to B.S., H. Y. W. or Y. H. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Song","suffix":""},{"id":400018852,"identity":"cd9e213b-ba95-4ba2-81d6-ef9a6a4d62f9","order_by":9,"name":"Houyu Wang","email":"","orcid":"https://orcid.org/0000-0002-5134-9881","institution":"Soochow university","correspondingAuthor":false,"prefix":"","firstName":"Houyu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-12-15 08:25:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5646556/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5646556/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73520047,"identity":"98c9e867-488a-4f0d-b00b-c6f0638e0e19","added_by":"auto","created_at":"2025-01-10 18:12:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16176551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic design and characterization of bacterial-specific ABC sugar transporter-responsive MR nanoprobes. a\u003c/strong\u003e, MT-MnHP nanoparticles synthesized by conjugating compound\u003cstrong\u003e 1\u003c/strong\u003e with \u003cstrong\u003e2\u003c/strong\u003e \u003cem\u003evia\u003c/em\u003e copper(I)-catalyzed click chemistry, followed by self-assembly into nanoparticles. \u003cstrong\u003eb\u003c/strong\u003e, Scheme showing the ABC sugar transporter enabling the selective delivery of magnetic resonance imaging nanoprobes into bacteria to visualize bacteria by shortening the T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e relaxation times, which enhances the light‒dark contrast in T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted images, respectively. \u003cstrong\u003ec\u003c/strong\u003e, TEM images and high-resolution TEM images of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. Scale bars, 20 nm or 1 nm. \u003cstrong\u003ed\u003c/strong\u003e, T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of MRSA treated with PBS, 0.3 mM MT-MnHPs, (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, MnHP, or maltotriose at 37 ℃ for 2 h. \u003cstrong\u003ee\u003c/strong\u003e, High-angle annular dark field-scanning TEM (HAADF-STEM) images of MDR \u003cem\u003eE. coli\u003c/em\u003e or MRSA treated with 0.3 mM MT-MnHPs, (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, MnHP, maltotriose, or PBS (control) at 37 ℃ for 2 h, with repeated rinses in PBS buffer following treatment (cell concentration ~1.0 × 10⁸ CFU). Scale bars, 500 nm or 200 nm. All imaging experiments were repeated three times with similar results.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/efa881848d85b7fbe4190f7b.png"},{"id":73520046,"identity":"7ef245e6-3703-4b9e-bbc9-aaf4bb309186","added_by":"auto","created_at":"2025-01-10 18:12:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16857928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterizations of bacterial-specific ABC sugar transporter-responsive MR nanoprobes in vitro. a\u003c/strong\u003e, UV‒visible absorption spectra of aqueous solutions of HP, MT-MnHPs, and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. \u003cstrong\u003eb\u003c/strong\u003e, Fluorescence emission spectra of HP, MT-MnHPs, and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs in water under 370 nm excitation. \u003cstrong\u003ec\u003c/strong\u003e, Time-resolved fluorescence emission spectra of SOSG (10 µM) with MT-MnHPs (0.3 mM) under ultrasound (US) exposure (1 MHz, 1.5 W cm⁻²). \u003cstrong\u003ed\u003c/strong\u003e, T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. \u003cstrong\u003ee\u003c/strong\u003e, Independent gradient model based on Hirshfeld partition (IGMH) calculations of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. \u003cstrong\u003ef\u003c/strong\u003e, 3D confocal laser scanning microscopy (CLSM) images and penetration profiles of MT-MnHPs and PBS in MRSA biofilms after 0, 1, and 2 min of treatment (scale bar, 20 µm). \u003cstrong\u003eg\u003c/strong\u003e, 3D CLSM images of untreated biofilms and biofilms after ultrasonic treatment for 2.5, 5, 7.5, and 10 min (scale bar, 50 µm). \u003cstrong\u003eh\u003c/strong\u003e, Crystal violet-stained images of MRSA and MDR \u003cem\u003eE. coli\u003c/em\u003e biofilms after various treatments. \u003cstrong\u003ei-j\u003c/strong\u003e, UV absorbance quantification of crystal violet-stained biofilms of MRSA (i) and MDR \u003cem\u003eE. coli\u003c/em\u003e (j) after different treatments (mean ± SD, n = 3). All imaging experiments were repeated three times with similar results. Statistical analysis was conducted \u003cem\u003evia\u003c/em\u003e paired two-tailed t tests. The error bars indicate the standard deviation (SD) from three independent measurements.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/59ad80b1468ea2507b6a2447.png"},{"id":73520368,"identity":"76a6c454-c1b5-4a11-ae28-5138aee77797","added_by":"auto","created_at":"2025-01-10 18:20:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8450237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial-specific ABC sugar transporter-responsive MR nanoprobes\u003c/strong\u003e \u003cstrong\u003etargeting diverse bacteria\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Confocal fluorescence images of MRSA and MDR \u003cem\u003eE. coli\u003c/em\u003e after incubation with MT-MnHPs (0.3 mM) and corresponding flow cytometry analysis of uptake rates. \u003cstrong\u003eb\u003c/strong\u003e, Confocal fluorescence images of the bacterial mutants ΔlamB and ΔmalE following incubation with MT-MnHPs (0.3 mM), with accompanying flow cytometry analysis of uptake efficiency. \u003cstrong\u003ec\u003c/strong\u003e, Confocal fluorescence images of MRSA and MDR \u003cem\u003eE. coli\u003c/em\u003e preincubated with various concentrations of maltotriose (0.3 mM and 0.6 mM) for 5 min, followed by incubation with MT-MnHPs (0.3 mM) for an additional 2 h (bacterial concentration ~10⁸ CFU). Scale bar, 25 µm. \u003cstrong\u003ed\u003c/strong\u003e, T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of PBS containing MRSA at various concentrations after treatment with MT-MnHPs (0.3 mM). \u003cstrong\u003ee\u003c/strong\u003e, Quantified T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities in PBS with MRSA at different concentrations after incubation with MT-MnHPs (0.3 mM) (mean ± SD, n = 3). \u003cstrong\u003ef\u003c/strong\u003e, T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of PBS containing MDR \u003cem\u003eE. coli\u003c/em\u003e at various concentrations after incubation with MT-MnHPs (0.3 mM) (mean ± SD, n = 3). \u003cstrong\u003eg\u003c/strong\u003e, T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities in PBS containing MDR \u003cem\u003eE. coli\u003c/em\u003e at different concentrations after incubation with MT-MnHPs (0.3 mM). All imaging experiments were repeated three times with similar results. Statistical analysis was conducted \u003cem\u003evia\u003c/em\u003e one-way ANOVA. The error bars indicate the standard deviation (SD) from three independent measurements.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/4a06fec097d615458f058a3c.png"},{"id":73520367,"identity":"0c4fefcf-3975-4dc5-88b2-1e184c1b0a12","added_by":"auto","created_at":"2025-01-10 18:20:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11642152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo imaging of nephritis in mouse models utilizing bacterial-specific ABC sugar transporter-responsive MR nanoprobes. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSchematic representation of MRI for MRSA-induced nephritis in mice employing Trojan MR probes. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eMRI of MRSA (~1.0 × 10⁸ CFU)-induced nephritis in mice subjected to various treatments as indicated. Infected mice received injections of PBS, HP, (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, or MT-MnHPs at equal doses.\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eCorresponding T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities for MRSA (~1.0 × 10⁸ CFU)-induced nephritis in mice under different treatment conditions (mean ± SD, n = 3). \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSchematic illustrating MRI for nephritis induced by 50% glycerin in mice using Gd-DTPA. \u003cstrong\u003ee\u003c/strong\u003e, T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of nephritis induced by PBS (control), 50% glycerin and MRSA (~1.0 × 10⁸ CFU) in mice utilizing MT-MnHPs. \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eT\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities for nephritis induced by PBS (control), 50% glycerin and MRSA (~1.0 × 10⁸ CFU) in mice treated with MT-MnHPs (mean ± SD, n = 3). \u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eT\u003csub\u003e1\u003c/sub\u003e-weighted MR images of nephritis induced by PBS (control), 50% glycerin and MRSA (~1.0 × 10⁸ CFU) in mice with Gd-DTPA.\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eT\u003csub\u003e1\u003c/sub\u003e signal intensities for nephritis induced by PBS (control), 50% glycerin and MRSA (~1.0 × 10⁸ CFU) in mice treated with Gd-DTPA (mean ± SD, n = 3). All imaging experiments were repeated three times with similar results. Statistical analysis was conducted \u003cem\u003evia\u003c/em\u003e one-way ANOVA. The error bars indicate the standard deviation (SD) from three independent measurements.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/8c15171cff01051e7ab17d19.png"},{"id":73520063,"identity":"9fb592c2-baf7-4d1b-8f79-de92b5686475","added_by":"auto","created_at":"2025-01-10 18:12:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8494754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo imaging of bacteria in tumours via bacterial-specific ABC sugar transporter-responsive MR nanoprobes. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSchematic representation of MRSA-infected sites and tumour sites (containing no MRSA) in mice treated with PBS or MT-MnHPs. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSchematic representation of MRSA-infected sites and tumour sites (containing MRSA) in mice treated with PBS or MT-MnHPs.\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eT\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of MRSA-infected sites and tumour sites (containing no MRSA).\u003cstrong\u003e d\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eT\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MR images of MRSA-infected sites and tumour sites (containing MRSA).\u003cstrong\u003e e\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eCorresponding T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities for two sites (containing no MRSA) (mean ± SD, n = 3). \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eCorresponding T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities for two sites (containing MRSA) (mean ± SD, n = 3). All imaging experiments were repeated three times with similar results. Statistical analysis was conducted \u003cem\u003evia\u003c/em\u003e paired two-tailed t tests. The error bars indicate the standard deviation (SD) from three independent measurements.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/296320be9b0df57bcffa3aff.png"},{"id":73520069,"identity":"71e39047-af79-4bd7-af6c-be12fe260320","added_by":"auto","created_at":"2025-01-10 18:12:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20984646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo antibacterial activity in mice with bacterial nephritis utilizing bacterial-specific ABC sugar transporter-responsive MR nanoprobes. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eSchematic representation of the Trojan MRI strategy for imaging and therapy in mice with bacterial nephritis.\u003cstrong\u003e b-c\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eHomogenates from infected kidneys of nephritis-bearing mice treated with PBS or MT-MnHPs, with or without ultrasound irradiation (1 MHz, 1.5 W cm⁻² for 10 min) seven days postinjection, cultured on solid LB agar (mean ± SD, n = 3 biologically independent samples) (b) and corresponding quantification of bacterial colonization (c). \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eHistological images of MRSA-infected kidney tissues from mice subjected to various treatments, as indicated. Scale bar, 25 μm. \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eMagnetic resonance imaging of nephritis-bearing mice at different treatment time points \u003cem\u003evia\u003c/em\u003e Trojan MR probes.\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eCorrelations between changes in T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal intensities over time and alterations in bacterial counts through quantitative analysis (mean ± SD, n = 3). All imaging experiments were performed in triplicate, yielding consistent results. Statistical analysis was conducted \u003cem\u003evia\u003c/em\u003e one-way ANOVA, with error bars representing standard deviations from three independent measurements.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/7cb2b2b330e7a09b7fd5507f.png"},{"id":75932887,"identity":"1161eb88-bfcf-4953-9220-4b7f56f34f0a","added_by":"auto","created_at":"2025-02-10 16:23:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76466358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/7d5e2cb3-dd5a-4081-8572-fea8697fd91d.pdf"},{"id":73520370,"identity":"3f490bf5-d1ac-4c76-a4e5-8d024db19483","added_by":"auto","created_at":"2025-01-10 18:20:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":44859983,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"Nat.Mater.BacteriatargetingMRIprobesSupporting.docx","url":"https://assets-eu.researchsquare.com/files/rs-5646556/v1/dae46124d9c0c90810be204b.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Selective magnetic resonance imaging of antibiotic-resistant bacteria leveraging ATP-binding cassette sugar transporter-responsive probes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) represents a pressing global challenge, with biofilm-associated infections exacerbating diagnostic complexities and ranking among the leading causes of mortality, potentially resulting in 10\u0026nbsp;million deaths by 2050\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. Current diagnostic approaches for bacterial infections rely primarily on blood tests and tissue biopsies, including bacterial culture, biochemical assays, immunoassays, polymerase chain reaction (PCR), and sequencing\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, these techniques are often labor intensive and time consuming. Additionally, invasive biopsies for deep tissue infections introduce risks, such as sampling errors, which can compromise diagnostic accuracy. Noninvasive imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), offer rapid, real-time and deep-tissue visualization of infections without the need for invasive procedures\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Despite these advantages, these techniques exhibit limited specificity, making distinguishing bacterial infections from nonbacterial inflammatory conditions, such as cancer or autoimmune disorders, challenging.\u003c/p\u003e \u003cp\u003eRecent advancements have introduced several targeted antimicrobial agents, including CRISPR‒Cas systems, engineered toxins, and stapled antimicrobial peptides\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Unlike these antimicrobial strategies, the \u0026ldquo;Trojan horse\u0026rdquo; antibiotic strategy\u0026mdash;originally proposed in the 1980s\u0026mdash;uses siderophore-linked antibiotics to exploit bacterial iron importers for intracellular delivery\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33 CR34 CR35\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This nutrient-based antibiotic delivery mechanism significantly mitigates bacterial resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, since these strategies still rely on antibiotics, the risk of resistance development persists. An alternative approach leverages bacteria-specific ATP-binding cassette (ABC) sugar transporters to transport photosensitizers into bacterial cells by coupling them with unique carbon sources, such as maltose, maltotriose, or maltohexose\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46 CR47 CR48\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Although these techniques enable both optical imaging and phototherapy for resistant infections, the limited tissue penetration of light (\u0026lt;\u0026thinsp;0.5 cm) restricts their application to superficial infections.\u003c/p\u003e \u003cp\u003eA more promising but underexplored strategy involves delivering multifunctional MR probes selectively into bacterial cells to detect and treat antibiotic-resistant infections deep within tissues. To fill this gap, we synthesized a series of bacterial-targeted MR nanoprobes consisting of azide-modified maltotriose (MT) linked to alkyne-modified manganese haematoporphyrin (HP) and self-assembled to form nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). As schematically illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, bacteria internalize the synthesized MR probes by recognizing maltotriose as a carbon source. Through ABC sugar transporters\u0026mdash;exemplified by \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e)\u0026mdash;maltotriose uptake occurs \u003cem\u003evia\u003c/em\u003e subunits such as LamB, MalE, MalF, MalG, and MalK. Specifically, LamB functions as an outer membrane porin, whereas MalE binds α (1\u0026ndash;4)-glucosidically linked maltotriose for transport\u003csup\u003e\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Analogously, our results confirm that various bacteria readily consume maltotriose-engineered MR probes disguised as nutrients. Upon internalization, manganese (Mn\u0026sup2;⁺) within these probes produces MR signals by shortening T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e relaxation times, enhancing both bright and dark contrast in T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted images, respectively. As the probes aggregate locally, they disrupt the magnetic field uniformity, accelerating transverse magnetization decay (M\u003csub\u003exy\u003c/sub\u003e) and generating a pronounced negative contrast effect\u003csup\u003e\u003cspan additionalcitationids=\"CR55 CR56 CR57\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In proof-of-concept studies, the developed MR nanoprobes enabled the detection of human-derived methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and multidrug-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e (MDR \u003cem\u003eE. coli\u003c/em\u003e) at concentrations as low as ~\u0026thinsp;10⁶ CFU. In addition to sensitive and specific imaging, haematoporphyrin in nanoprobes demonstrates ultrasound-activated antimicrobial activity, underscoring its potential to selectively target and treat bacterial infections in deep tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDesign of bacteria-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe synthesized two bacterial-specific ABC sugar transporter-responsive MR nanoprobes, i.e., MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, the detailed synthesis of which is described in the \u003cb\u003eSupplementary Methods\u003c/b\u003e. Typically, MT-MnHP is a manganese-based haematoporphyrin compound featuring a tetramethylporphyrin core functionalized with a single maltoriose-modified triazole linker. (MT)₂-MnHP shares the tetramethylporphyrin core of MT-MnHP but includes a dual maltoriose-modified triazole linker. Briefly, an azide-functionalized maltotriose intermediate (compound \u003cb\u003e1\u003c/b\u003e) was synthesized at the anomeric carbon to facilitate subsequent functionalization \u003cem\u003evia\u003c/em\u003e copper(I)-catalyzed click chemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This intermediate was prepared following a modified protocol\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplementary Figs.\u0026nbsp;1, 5 \u0026amp; 6\u003c/b\u003e). In parallel, alkyne-functionalized hematoporphyrins (compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e) were obtained \u003cem\u003evia\u003c/em\u003e Williamson ether synthesis (\u003cb\u003eSupplementary Figs.\u0026nbsp;2, 7 \u0026amp; 8\u003c/b\u003e) and further chelated with manganese ions through a thermal solvent approach (\u003cb\u003eSupplementary Figs.\u0026nbsp;2, 9 \u0026amp; 10\u003c/b\u003e). These alkyne-derivatized manganese porphyrins were conjugated to the azide-functionalized maltotriose intermediate \u003cem\u003evia\u003c/em\u003e click chemistry (\u003cb\u003eSupplementary Figs.\u0026nbsp;3 \u0026amp; 4\u003c/b\u003e). Through self-assembly, the final bacteria-targeting MR nanoprobes, MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, were successfully synthesized.\u003c/p\u003e \u003cp\u003eScanning transmission electron microscopy (STEM) images revealed that the synthesized nanoprobes exhibited a spherical morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The hydrodynamic diameters of the MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, as measured by dynamic light scattering (DLS), were approximately 3.4 nm and 8.3 nm, respectively (\u003cb\u003eSupplementary Fig.\u0026nbsp;11\u003c/b\u003e). The slight discrepancy in particle size between DLS and TEM likely reflects differences in surface conditions under the two measurement techniques. Elemental mapping (\u003cb\u003eSupplementary Fig.\u0026nbsp;12\u003c/b\u003e) confirmed the uniform distribution of Mn, C, N, and O within the particles. While the manganese signals corresponded to the coordinated Mn ions, the carbon, nitrogen, and oxygen signals originated from the hematoporphyrin and maltotriose linkers. These results confirm the successful synthesis and nanoparticle formation of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs.\u003c/p\u003e \u003cp\u003eTo investigate the ability of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs to penetrate bacterial cells, MDR \u003cem\u003eE. coli\u003c/em\u003e and MRSA were isolated from keratitis patients treated at the Shanghai Eye, Ear, Nose, and Throat Hospital, Fudan University. The isolated strains (~\u0026thinsp;1.0 \u0026times; 10⁸ CFU) were incubated with 0.3 mM solutions of MT-MnHPs or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs at 37 ℃ for 2 hours, followed by several washes with phosphate-buffered saline (PBS). High-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) and elemental mapping revealed that while C, N, and O were detected in all bacterial samples, Mn signals were present exclusively in the groups treated with MT-MnHPs or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). These results confirmed the internalization of the Trojan MR probes into bacterial cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEx vivo\u003c/em\u003e MRI scans of the treated bacterial samples were performed \u003cem\u003evia\u003c/em\u003e a 3.0 T clinical MRI scanner with fast spin‒echo T\u003csub\u003e1\u003c/sub\u003e-weighted imaging (FSE T\u003csub\u003e1\u003c/sub\u003eWI) and T\u003csub\u003e2\u003c/sub\u003e-weighted imaging (FSE T\u003csub\u003e2\u003c/sub\u003eWI). A T\u003csub\u003e1\u003c/sub\u003e signal enhancement or T\u003csub\u003e2\u003c/sub\u003e signal decrease was observed exclusively in the bacteria treated with MT-MnHPs or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, with approximately twofold stronger signals than those in the untreated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cb\u003eSupplementary Figs.\u0026nbsp;13 \u0026amp; 14\u003c/b\u003e). Notably, the MR images exhibited differential behavior: T\u003csub\u003e1\u003c/sub\u003e-weighted images displayed brighter signals, whereas T\u003csub\u003e2\u003c/sub\u003e-weighted images gradually darkened (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These findings demonstrate that MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs function effectively as dual-mode MR probes and can successfully infiltrate bacterial cells, establishing their utility for T\u003csub\u003e1\u003c/sub\u003e/T\u003csub\u003e2\u003c/sub\u003e-weighted imaging applications.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro assessment of bacterial-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e The synthesized MR probes employed Mn(II) ions as the central components, which act as key MRI indicators. UV\u0026ndash;vis absorption and fluorescence (FL) spectra were utilized to explore the interactions between Mn(II) ions and HP within both MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. The absorption spectrum of HP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) displayed a characteristic Soret band (393 nm) and four Q-bands (496, 531, 566, and 619 nm), corresponding to electron transitions from the HOMO to the π* orbitals. While both probes retained these typical bands, MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs also exhibited Soret peaks at 368 or 371 nm, along with two Q-bands spanning 540\u0026ndash;600 nm, and introduced a novel peak at 460 nm. The emergence of these two Q-bands confirmed the successful coordination of manganese ions to the porphyrin core, where two Q-bands resulted from π-to-π* transitions within the aromatic porphyrin framework.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder 370 nm light excitation, both probes presented two weak fluorescence peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), with lower intensities than those of pure HP, indicating energy transfer from HP to Mn ions and maltotriose. A higher maltotriose content increased energy transfer, confirming that Mn(II) ions selectively coordinate with the porphyrin ring and that MT-MnHPs exhibited superior optical activity relative to that of (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. The impact of maltose linkages on sonodynamic therapy (SDT) efficiency was investigated by measuring singlet oxygen (\u0026sup1;O₂) production via SOSG. MT-MnHPs generated \u0026sup1;O₂ under ultrasound (US) irradiation, with the yield increasing over time and reaching a plateau after 10 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This plateau established 10 minutes as the optimal excitation period. Notably, MT-MnHPs outperformed (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs in generating \u0026sup1;O₂ (\u003cb\u003eSupplementary Fig.\u0026nbsp;15\u003c/b\u003e), suggesting that differences in maltose linkages may influence ROS generation efficiency. The sonotoxicity of MT-MnHPs against MRSA was assessed \u003cem\u003evia\u003c/em\u003e a turbidity assay following 10 minutes of US exposure. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;16\u003c/b\u003e, bacterial inhibition correlated with increasing concentrations of HP, MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. At 0.3 mM, MT-MnHPs completely inhibited bacterial growth, whereas HP and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs achieved only 29% and 67% inhibition, respectively. Although both probes produced ROS under US, HP failed to penetrate bacteria, and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs demonstrated weaker ROS production, limiting their antibacterial efficacy.\u003c/p\u003e \u003cp\u003eMotivated by the successful coordination of Mn ions to the porphyrin core, we next examined the MRI contrast properties. As the concentration of either MT-MnHPs or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs increased from 0 to 0.3 mM, the T\u003csub\u003e1\u003c/sub\u003e-weighted MR images brightened, whereas the T\u003csub\u003e2\u003c/sub\u003e-weighted images dimmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). This dual contrast behavior was corroborated by corresponding signal intensity changes, with increasing T\u003csub\u003e1\u003c/sub\u003e signals and decreasing T\u003csub\u003e2\u003c/sub\u003e signals (\u003cb\u003eSupplementary Fig.\u0026nbsp;17\u003c/b\u003e). In addition, MT-MnHPs showed a marked MRI contrast with a relaxivity r\u003csub\u003e1\u003c/sub\u003e as high as 11.56 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;18a\u003c/b\u003e), which is almost 3.0-fold greater than that of (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. In addition, the r\u003csub\u003e2\u003c/sub\u003e value of MT-MnHPs is 102.0 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;18b\u003c/b\u003e), which is significantly greater than that of (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. Additionally, changes in the signal-to-noise ratios (ΔSNRs) were obtained by quantifying the differences in signal enhancements in the images in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed for a better comparison of the MRI performance of the NPs. The ΔSNR of MT-MnHPs was much higher than that of (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs at all concentrations above 0.075 mM (\u003cb\u003eSupplementary Fig.\u0026nbsp;18c\u003c/b\u003e), which further indicates the significantly enhanced MRI performance of MT-MnHPs.\u003c/p\u003e \u003cp\u003eProton\u0026ndash;proton or proton\u0026ndash;electron interactions involving water protons and paramagnetic ions or molecules predominantly govern the functionality of paramagnetic centres as contrast agents\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. To elucidate the molecular-level MRI contrast characteristics of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, density functional theory (DFT) calculations were conducted \u003cem\u003evia\u003c/em\u003e the b3lyp/def2SVP functional\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Additionally, the independent gradient model based on Hirshfeld partitioning (IGMH) was applied through the Multiwfn program\u003csup\u003e\u003cspan additionalcitationids=\"CR64 CR65\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The analysis revealed the presence of intermolecular forces, including attractive, repulsive, and van der Waals interactions, between water molecules and MT ligands in MT-MnHPs or (MT)₂-MnHPs, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee (I and II). Both MT-MnHPs and (MT)₂-MnHPs exhibited weak noncovalent interactions (green arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), which facilitated the enrichment of bound water molecules near the paramagnetic centers. This binding restricts the movement of water molecules, thereby mitigating the averaging effect and shortening their relaxation times. Furthermore, the binding energy between water and MT ligands in MT-MnHPs (79.8 kJ/mol) exceeds that in (MT)₂-MnHPs (73.2 kJ/mol), underscoring the superior MRI contrast performance of MT-MnHPs.\u003c/p\u003e \u003cp\u003eMost conventional treatments fail to eliminate biofilms because of limited bactericidal penetration. Given the demonstrated bacterial targeting ability and superior sonodynamic activity of MT-MnHPs, we investigated their potential to disrupt biofilms. MRSA biofilms served as the model for this proof-of-concept study. Using FITC-labelled biofilms, we observed that MT-MnHPs combined with US generated red fluorescence at the depth of penetration after 2 minutes of US exposure, indicating efficient penetration driven by bacterial uptake and ultrasound-assisted diffusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Crystal violet staining was used to quantify the sonodynamic effect on biofilm integrity. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, untreated biofilms remained thick and structurally intact, whereas those treated with MT-MnHPs and subjected to increasing US irradiation times presented significant reductions in thickness and cohesion. Quantitative analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej and \u003cb\u003eSupplementary Fig.\u0026nbsp;19\u003c/b\u003e) revealed that MT-MnHPs\u0026thinsp;+\u0026thinsp;US eradicated 97% of the MRSA biofilms, outperforming (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs\u0026thinsp;+\u0026thinsp;US, which cleared 78% of the MRSA biofilms. Similarly, compared with (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, MT-MnHPs achieved 97% clearance of MDR \u003cem\u003eE. coli\u003c/em\u003e biofilms, whereas (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs achieved 80% clearance. These findings establish MT-MnHPs as effective probes for sonodynamic biofilm eradication.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial-specific ABC sugar transporter-responsive MR nanoprobes target diverse bacterial species.\u003c/b\u003e To optimize the incubation time, we systematically investigated the uptake efficiency of MT-MnHPs (0.3 mM) by bacteria through flow cytometry. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;20\u003c/b\u003e, the uptake of ~\u0026thinsp;1.0 \u0026times; 10⁸ CFU of MRSA and MDR \u003cem\u003eE. coli\u003c/em\u003e reached maximum levels\u0026mdash;73.7% for MRSA and 62.4% for MDR \u003cem\u003eE. coli\u003c/em\u003e\u0026mdash;after 2 hours of incubation. Extending the incubation period yielded no significant increase in uptake, indicating that saturation had been achieved. Therefore, all following experiments were performed after incubating with MT-MnHPs (0.3 mM) for 2 h.\u003c/p\u003e \u003cp\u003eNext, we assessed the ability of MT-MnHPs to target a range of natural bacterial strains. We selected two gram-negative species\u0026mdash;MDR \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella typhimurium\u003c/em\u003e (STm)\u0026mdash;and two gram-positive species\u0026mdash;MRSA and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e). Confocal laser scanning microscopy (CLSM) images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cb\u003eSupplementary Fig.\u0026nbsp;21\u003c/b\u003e) revealed prominent red fluorescence signals from MT-MnHPs (emission: 600\u0026ndash;700 nm, excitation: 370 nm) in all four bacterial strains after incubation with MT-MnHPs (0.3 mM) for 2 hours. Quantitative analysis \u003cem\u003evia\u003c/em\u003e flow cytometry confirmed uptake efficiencies of 73.7% for MRSA, 62.4% for MDR \u003cem\u003eE. coli\u003c/em\u003e, 64.9% for \u003cem\u003eS. aureus\u003c/em\u003e, and 72.5% for STm. In contrast, negligible fluorescence was observed when bacteria were treated with unmodified HP (\u003cb\u003eSupplementary Fig.\u0026nbsp;22\u003c/b\u003e), confirming that maltotriose modification is essential for bacterial targeting.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further explored whether MT-MnHPs rely on ABC sugar transporters for bacterial uptake. Two bacterial mutants\u0026mdash;ΔlamB and ΔmalE\u0026mdash;were generated and validated through Sanger sequencing (\u003cb\u003eSupplementary Notes\u003c/b\u003e). As anticipated, no fluorescence signals were detected in the ΔlamB or ΔmalE mutants treated with MT-MnHPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), which is consistent with the CLSM observations. Additionally, competition assays demonstrated that preincubation of bacteria with increasing concentrations of maltotriose (0, 0.3, or 0.6 mM) for 5 min significantly diminished MT-MnHP uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These findings confirm that MT-MnHPs enter bacteria through the ABC sugar transporter pathway.\u003c/p\u003e \u003cp\u003eWe next assessed the specificity of MT-MnHPs for bacteria over mammalian cells. HeLa cells and human blood samples spiked with MRSA were treated with MT-MnHPs (0.3 mM) for 2 hours and washed with PBS. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;23\u003c/b\u003e, red fluorescence was detected only in bacterial cells, with no signal observed in HeLa cells or blood cells, indicating minimal uptake by mammalian cells. This selectivity arises from the absence of ABC sugar transporters in mammalian cells. To determine the detection limit, we imaged serial dilutions of MRSA and MDR \u003cem\u003eE. coli\u003c/em\u003e incubated with the MR probes (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Bacterial concentrations as low as ~\u0026thinsp;10⁶ CFU produced detectable signals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), highlighting the sensitivity of the approach. These results demonstrate the potential of bacteria-targeting MR nanoprobes for clinical bacterial diagnostics.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMagnetic resonance imaging of bacterial nephritis in mice via bacterial-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e We evaluated the feasibility of the developed MR nanoprobes for imaging bacteria residing deep within tissues. To establish a proof-of-concept model, we induced nephritis in mice \u003cem\u003evia\u003c/em\u003e MRSA infection. As shown schematically in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, female mice (6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;3) were injected with 25 \u0026micro;L of MRSA, followed 12 hours later by intravenous administration of 200 \u0026micro;L of PBS, HP (0.3 mM), MT-MnHPs (0.3 mM), or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (0.3 mM). The concentration of MRSA at the infection site, measured by harvesting and culturing kidney tissue, was ~\u0026thinsp;1.0 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU. We observed that the strongest MR signal was localized exclusively at the infected site in the mice treated with MT-MnHPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Quantitative analysis revealed that the T\u003csub\u003e1\u003c/sub\u003e or T\u003csub\u003e2\u003c/sub\u003e signal intensities in the MT-MnHP-treated group were approximately twice those observed in the (MT)\u003csub\u003e2\u003c/sub\u003e-MnHP-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Notably, the developed MR nanoprobes demonstrated the ability to detect bacterial concentrations as low as ~\u0026thinsp;1.0 \u0026times; 10⁶ CFU within the kidney (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;24\u003c/b\u003e\u0026mdash;sensitivity adequate for many in vivo scenarios.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the specificity of the developed MR nanoprobes for bacterial nephritis, we developed a comparative model of glycerin-induced nephritis. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, female mice (6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;3) were injected in situ with 25 \u0026micro;L of 50% (v/v) glycerin, followed by an intravenous injection of 200 \u0026micro;L of MT-MnHPs (0.3 mM) 12 hours later. As expected, T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e signal changes were significantly greater in the mice with MRSA nephritis than in those with glycerin-induced nephritis or in the control mice treated with MT-MnHPs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eFurthermore, we compared the imaging performance of the developed MR nanoprobes with that of gadopentetic acid (Gd-DTPA), a clinically used contrast agent. Both MRSA-infected and glycerin-nephritis-bearing mice were scanned with a 3.0 T MRI scanner \u003cem\u003evia\u003c/em\u003e a T\u003csub\u003e1\u003c/sub\u003e-weighted sequence (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Consistent with prior reports\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, Gd-DTPA exhibited strong T\u003csub\u003e1\u003c/sub\u003e signal enhancement due to the presence of Gd\u0026sup3;⁺, which promoted longitudinal proton relaxation through unpaired electrons and extended the electron spin relaxation time. However, unlike the bacterial-specific ABC sugar transporter-responsive MR nanoprobes, which selectively enhanced signals only at sites of bacterial nephritis, Gd-DTPA increased T\u003csub\u003e1\u003c/sub\u003e signals in both the bacterial and nonbacterial nephritis models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). These results underscore the high selectivity of the developed MR nanoprobes, which can effectively distinguish bacterial nephritis from other inflammatory conditions. The specificity and tunable sensitivity of the developed method are attributed to the preferential internalization of the developed MR nanoprobes within bacterial cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMagnetic resonance imaging of bacteria in tumours via bacterial-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e Next, we verified the effectiveness of the developed MR nanoprobes for imaging bacteria in tumours. Accordingly, we constructed proof-of-concept models of bacteria in tumour xenografts. To construct the tumour xenograft model, we subcutaneously injected 100 \u0026micro;L of CT26 cells (~\u0026thinsp;5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) into the right back region of female nude mice (6\u0026ndash;8 weeks old). When the tumours grew to 100 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, we subcutaneously injected 50 \u0026micro;L of MRSA into the left thigh region of the mice or into both the left thigh region and the right tumour region of the mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), followed by intravenous injection of 200 \u0026micro;L of PBS or MT-MnHPs (0.3 mM). The infected sites as well as the tumour sites were then imaged by a 3.0 T MRI scanner using a T\u003csub\u003e1\u003c/sub\u003e-weighted sequence and T\u003csub\u003e1\u003c/sub\u003e-weighted sequence at 6 h postinjection of MT-MnHPs (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). As revealed in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, we observed MR signals only at the infected sites instead of at the tumour sites containing no bacteria, indicating that the developed MR nanoprobes enabled the discrimination of bacteria from tumors. As expected, the detection signals from the infected sites treated with MT-MnHPs were significantly stronger than those from their counterparts treated with PBS were (e.g., ~\u0026thinsp;2.0 (MRSA, left)-fold increase in T\u003csub\u003e1\u003c/sub\u003e-weighted signals and ~\u0026thinsp;2.1 (MRSA, left)-fold decrease in T\u003csub\u003e2\u003c/sub\u003e-weighted signals) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). As further revealed in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, we observed MR signals simultaneously at the infected sites and the tumor sites containing bacteria. Consistently, the signals detected from both the infected sites and the tumour sites containing bacteria treated with MT-MnHPs were much stronger than those detected with PBS (e.g., ~\u0026thinsp;2.7 (MRSA, left) and ~\u0026thinsp;2.1 (tumor\u0026thinsp;+\u0026thinsp;MRSA, right)-fold increase in T\u003csub\u003e1\u003c/sub\u003e-weighted signals and ~\u0026thinsp;1.8 (MRSA, left) and ~\u0026thinsp;1.8 (tumor\u0026thinsp;+\u0026thinsp;MRSA, right)-fold decrease in T\u003csub\u003e2\u003c/sub\u003e-weighted signals). These results together prove that the developed MR nanoprobes allowed the imaging of bacteria residing within tumour tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro antibacterial activity of the developed strategy.\u003c/b\u003e In addition to enabling MRI of bacterial infections, the synthesized MT-MnHPs (0.3 mM) exhibited potent antibacterial effects due to their sonodynamic properties. We verified this antimicrobial activity through a live/dead bacterial staining assay. In this assay, the green fluorescent dye NO 1 (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;488 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;525 nm) marked both intact (viable) and damaged (nonviable) bacterial membranes, whereas propidium iodide (PI, λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;536 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;617 nm) selectively penetrated compromised membranes, emitting red fluorescence to indicate bacterial cell death. As demonstrated in \u003cb\u003eSupplementary Fig.\u0026nbsp;25\u003c/b\u003e, green fluorescence dominated in the control groups, whereas treatment with MT-MnHPs or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs under SDT elicited increased red fluorescence, confirming bacterial membrane disruption.\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) images provided further morphological evidence of bacterial damage (\u003cb\u003eSupplementary Fig.\u0026nbsp;26\u003c/b\u003e). MRSA and MDR \u003cem\u003eE. coli\u003c/em\u003e cells incubated with MT-MnHPs (0.3 mM) for 2 hours, followed by ultrasound irradiation (1 MHz, 1.5 W/cm\u0026sup2;) for 10 minutes, displayed wrinkling or lysis of the cell membranes. These effects were more pronounced with MT-MnHPs than with (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, aligning with their superior singlet oxygen (\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO₂) generation, which enhanced the SDT efficacy.\u003c/p\u003e \u003cp\u003eWe next investigated the penetration depth of ultrasound in modulating the antibacterial efficiency of MT-MnHPs. Using chicken breast tissues of varying thicknesses (0 to 4.0 cm) placed over bacterial cultures in 24-well plates (\u003cb\u003eSupplementary Fig.\u0026nbsp;27b\u003c/b\u003e), we performed agar plate experiments following ultrasound irradiation (1 MHz, 1.5 W/cm\u0026sup2;) for 10 minutes. As shown in \u003cb\u003eSupplementary Figs.\u0026nbsp;27a-27c\u003c/b\u003e, the antibacterial efficacy decreased as the tissue thickness increased. When the tissue was 4.0 cm thick, the antibacterial rates were approximately 65% for MRSA and 61% for MDR \u003cem\u003eE. coli\u003c/em\u003e. In contrast, the PBS-treated groups showed no antibacterial effects with increasing tissue thickness. These results highlight the robust penetration depth of MT-MnHPs, establishing a strong basis for future applications of SDT in deep tissues. The superior sonodynamic performance of MT-MnHPs compared with that of (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs underscores their potential for in vivo therapeutic interventions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo antibacterial activity of the developed strategy.\u003c/b\u003e To assess the in vivo antibacterial efficacy of the developed strategy, we evaluated its performance in a proof-of-concept bacterial nephritis model in mice. As illustrated schematically in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, MRSA (1.0 \u0026times; 10⁸ CFU) was injected intravenously into female mice (6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;3). At 12 hours post infection, the mice received 200 \u0026micro;L of MT-MnHPs (0.3 mM) or PBS through intravenous injection on days 1, 3, 5, and 7. Four hours after each injection, we applied US to initiate SDT, with each session lasting 10 minutes. Imaging was performed following each irradiation to monitor therapeutic progress in real time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePosttreatment, infected kidney tissues were excised, homogenized, and cultured on agar plates to quantify the bacterial load. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb \u0026amp; \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, only a few colonies remained in the MT-MnHPs\u0026thinsp;+\u0026thinsp;US group after seven days of treatment. The antibacterial rate of MT-MnHPs under ultrasound reached 96% against MRSA. Consistent with these findings, hematoxylin‒eosin (H\u0026amp;E) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed) revealed intact tissue architecture and the absence of cell necrosis exclusively in the MT-MnHPs\u0026thinsp;+\u0026thinsp;US group. This outcome highlights the therapeutic benefit of SDT in achieving high antibacterial efficacy.\u003c/p\u003e \u003cp\u003eAdditionally, the developed strategy enabled real-time visualization of the antibacterial effects. MRI was performed after each irradiation session to assess therapeutic outcomes dynamically. As depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee \u0026amp; \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, the MR signal gradually diminished as the treatment progressed, which was correlated with reductions in the bacterial load (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Quantitative analysis confirmed that the decrease in MR signal intensity was directly proportional to bacterial clearance. This alignment between MR signal changes and bacterial counts underscores the utility of bacterial concentration-dependent MRI as a reliable marker for monitoring both detection and treatment efficacy. Together, these data validate the adaptive antibacterial potential of this strategy in vivo, demonstrating its feasibility for precise and effective therapeutic applications.\u003c/p\u003e \u003cp\u003e \u003cb\u003eToxicity assessment of bacterial-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e We comprehensively evaluated the toxicity profiles of MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;28a\u003c/b\u003e, methyl thiazolyl tetrazolium (MTT) assays revealed that the viability of CT26, mREC, HeLa, and MCF-7 cells exceeded 80% following incubation with either MT-MnHPs (0.3 mM) or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (0.3 mM). These findings indicate that both probes exhibit minimal cytotoxicity under the tested conditions.\u003c/p\u003e \u003cp\u003eWe further assessed hemolytic activity by exposing red blood cells to MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs at concentrations up to 0.3 mM. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;28b\u003c/b\u003e, the hemolysis rates were 9.8% for MT-MnHPs and 10.8% for (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs, indicating favourable hemocompatibility. Additionally, H\u0026amp;E staining of major organs (\u003cb\u003eSupplementary Fig.\u0026nbsp;28c\u003c/b\u003e) harvested 24 hours postinjection revealed no histopathological abnormalities, further confirming the negligible in vivo toxicity of both probes.\u003c/p\u003e \u003cp\u003eTo assess systemic toxicity, we conducted blood biochemistry and routine hematological analyses at the administered dose. As shown in \u003cb\u003eSupplementary Figs.\u0026nbsp;29a-29h\u003c/b\u003e, all biochemical parameters for the MT-MnHP and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHP groups remained within normal ranges and were comparable to those of the PBS control group. \u003cem\u003eEx vivo\u003c/em\u003e fluorescence imaging (\u003cb\u003eSupplementary Fig.\u0026nbsp;30a\u003c/b\u003e) further revealed that the probes predominantly accumulated in the liver and kidneys at 0.5 hours postinjection, with negligible fluorescence signals detected in other organs. Importantly, the fluorescence signals had almost entirely disappeared by 48 hours, suggesting efficient clearance. Furthermore, strong fluorescence signals were detected in urine samples collected 24 hours postinjection (\u003cb\u003eSupplementary Fig.\u0026nbsp;30b\u003c/b\u003e), confirming renal excretion as a primary elimination route. These results collectively demonstrate that MT-MnHPs and (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs exhibit minimal toxicity, high biocompatibility, and effective clearance, supporting their potential for in vivo applications.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ABC sugar transporter-responsive MR nanoprobes presented here demonstrate a potent and selective approach for diagnosing and treating human-derived antibiotic-resistant bacterial infections in deep tissues. By harnessing maltotriose-coupled manganese porphyrin derivatives, we enabled selective bacterial targeting through ABC sugar transporters, facilitating robust MR contrast and ultrasound-activated antimicrobial activity. This bacterial-specific uptake mechanism enables visualization of bacterial distribution within deep tissues and has potential for combined diagnostic and therapeutic applications. Our findings address the long-standing limitation of the poor specificity of conventional MRI for bacterial infections.\u003c/p\u003e \u003cp\u003eDespite these promising outcomes, several limitations warrant consideration. First, while in vitro and murine models have validated the selective uptake and therapeutic efficacy of these MR probes, further studies are needed to verify their effectiveness and safety in larger animal models and human tissues. The bacterial strains tested, although representative, do not cover the full spectrum of clinically relevant pathogens, and variation in ABC sugar transporter expression across bacterial species may influence probe uptake. Additionally, while ultrasound-activated antimicrobial effects have potential, the depth of ultrasound penetration may limit its therapeutic efficacy in larger or denser tissues. To optimize this strategy for clinical applications, future studies should evaluate the safety profile and pharmacokinetics of these agents across a range of bacterial infections and tissue types. Enhancing probe specificity and retention within target tissues, refining ultrasound parameters to maximize tissue penetration, and developing real-time imaging protocols could further strengthen the translational potential of this approach.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePreparation and purification of bacterial-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRefer to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. MT-MnHPs and (MT)₂-MnHPs were prepared by coupling alkyne-functionalized manganese porphyrins (compound \u003cb\u003e2\u003c/b\u003e) with azide-functionalized maltotriose (compound \u003cb\u003e1\u003c/b\u003e) through a copper-catalyzed azide‒alkyne cycloaddition (click reaction). The detailed synthesis protocols and characterizations of intermediates \u003cb\u003e1\u003c/b\u003e, \u003cb\u003e2\u003c/b\u003e, and \u003cb\u003e3\u003c/b\u003e are provided in the Supplementary Information. The specific click reaction between \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e to generate MT-MnHPs is described as follows: Compound \u003cb\u003e1\u003c/b\u003e (50 mg, 0.05 mmol) and compound \u003cb\u003e2\u003c/b\u003e (69 mg, 0.10 mmol) were dissolved in DMF (10 mL). CuI (0.2 mg, 1.0 \u0026micro;mol) and DIPEA (1.2 mg, 0.01 mmol) were then added to the solution. The reaction mixture was stirred at ambient temperature under nitrogen for 12 hours. The solvent was removed under reduced pressure, and the residue was dissolved in CH₂Cl₂ (30 mL). The organic phase was washed sequentially with water (10 mL) and saltwater (10 mL), dried over Na₂SO₄, filtered, and evaporated to dryness under vacuum. The resulting acetyl-protected intermediate was deprotected in a mixture of CH₃OH (2 mL) and aqueous LiOH (1.0 M, 2 mL) under nitrogen for 24 hours. The crude (MT)₂-MnHP were neutralized \u003cem\u003evia\u003c/em\u003e Dowex 50 W resin, filtered, and concentrated under vacuum. MT-MnHP was further purified \u003cem\u003evia\u003c/em\u003e silica gel flash chromatography. To obtain MT-MnHP nanoparticles, 10 mg of MT-MnHP was dissolved in 1 mL of DMSO and added dropwise to 9 mL of deionized water under sonication with magnetic stirring. The suspension was stirred overnight, followed by centrifugation (10,000 rpm, 10 min) and washing with deionized water. A detailed characterization of MT-MnHPs and the synthesis and characterization of (MT)₂-MnHPs can be found in the Supplementary Information.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterizations of bacterial-specific ABC sugar transporter-responsive MR nanoprobes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the morphology and size of bacteria-targeting MR probes \u003cem\u003evia\u003c/em\u003e transmission electron microscopy (TEM) at 200 kV (Philips CM 200). UV‒visible (UV‒vis) absorption spectra were captured on a Perkin-Elmer Lambda 750 UV‒vis/NIR spectrophotometer. Photoluminescence spectra were recorded \u003cem\u003evia\u003c/em\u003e a HORIBA Fluoromax-4 spectrofluorometer. Dynamic light scattering (DLS) measurements were taken with a Delsa\u0026trade; Nano submicron particle analyser. For bacterial fluorescence imaging in vitro, we used a Leica TCS-SP5 II confocal laser scanning microscope (CLSM). Ex vivo and in vivo MR images were acquired with a 3.0 \u003cem\u003eT\u003c/em\u003e clinical MRI scanner utilizing fast spin echo (FSE) T\u003csub\u003e1\u003c/sub\u003e-weighted (T\u003csub\u003e1\u003c/sub\u003eWI) and T\u003csub\u003e2\u003c/sub\u003e-weighted (T\u003csub\u003e2\u003c/sub\u003eWI) sequences.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial culture.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMultidrug-resistant (MDR) \u003cem\u003eE. coli\u003c/em\u003e and methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), which were isolated from keratitis patients, were obtained from the Eye, Ear, Nose and Throat Hospital, Fudan University, with ethical approval (EENTIRB-2017-06-07-01). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eSalmonella typhimurium\u003c/em\u003e (BNCC 108207) were sourced from the American Type Culture Collection and BeNa Culture Collection, respectively. Bacteria were cultured in Luria\u0026ndash;Bertani (LB) medium, initially rehydrated in liquid LB, plated on LB agar, and incubated at 37 ℃ for 12 hours. A single colony was picked and cultured in LB medium at 250 rpm and 37 ℃. Bacterial cells at exponential growth were collected, and concentrations were confirmed by optical density at 600 nm. Colony numbers were quantified with a colony counter (Czone 8). The protocols conformed to the Declaration of Helsinki and Chinese regulations.\u003c/p\u003e\n\u003ch3\u003eMinimum inhibitory concentration (MIC) of MT-MnHPs\u003c/h3\u003e\n\u003cp\u003eMDR \u003cem\u003eE. coli\u003c/em\u003e and MRSA were cultured in LB media at 37 ℃ on a shaker (200 rpm) for 4\u0026ndash;6 hours. The bacterial concentrations were adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.1 for 1 \u0026times; 10⁸ CFU/mL. Bacterial suspensions (20 \u0026micro;L of 1 \u0026times; 10⁹ CFU/mL) were added to PBS (160 \u0026micro;L per well). Phosphate-buffered saline (PBS), HP, MT-MnHPs, or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs were added to a Corning 96-well plate and incubated at 37 ℃ and 200 rpm for 18 hours. OD\u003csub\u003e600\u003c/sub\u003e readings were recorded with a Tecan Infinite M200 microplate reader, with triplicate cultures for each assay, which was repeated at least twice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro cellular experiments.\u003c/b\u003e A mouse colorectal cancer cell line (CT26 cells) and human cervical cells (HeLa cells) were cultured in 1640 medium. Human breast cancer cells (MCF-7 cells) and mouse retinal endothelial cells (mRECs), cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium with high glucose (H-DMEM), were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (China). All the abovementioned media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% relevant antibiotics (100 \u0026micro;g/mL streptomycin and 100 U/mL penicillin). All the cell lines were cultured at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator with a humidified atmosphere.\u003c/p\u003e\n\u003ch3\u003eHuman studies\u003c/h3\u003e\n\u003cp\u003e Blood samples were obtained from a healthy donor who provided written informed consent. All procedures involving human blood were conducted with approval from Soochow University's ethics committee in compliance with institutional and legal standards. Vitreous fluid samples (0.1 mL, nondiluted and sterile) were drawn from ten patients diagnosed with bacterial endophthalmitis during diagnostic pars plana vitrectomy (PPV) procedures. These samples were collected \u003cem\u003evia\u003c/em\u003e a 30 G needle and transferred immediately to presterilized microfuge tubes for imaging. Additional clinical samples were provided by the Eye Bank at the Eye, Ear, Nose, and Throat Hospital, Fudan University, with ethics committee approval (EENTIRB-2017-06-07-01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCLSM 3D imaging and crystal violet staining of biofilms.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMRSA, diluted to 10⁵ CFU/mL (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.025), was added (120 \u0026micro;L) to 96-well plates and cultured at 37 ℃ for 24 hours. After washing with PBS, mature biofilms were treated with PBS or MT-MnHPs (0.3 mM), followed by ultrasound exposure (1 MHz, 1.5 W/cm\u0026sup2;, 0\u0026ndash;2 min). The biofilms were fixed in 4% glutaraldehyde for 4 hours, stained with ethidium bromide (EB) and FITC-ConA at 4 ℃ for 15 minutes, and observed \u003cem\u003evia\u003c/em\u003e CLSM 3D imaging. Mature biofilms were prepared as described above. A 2 mL aliquot of bacterial suspension was added to a 12-well growth plate containing sterile coverslips positioned vertically and incubated under static conditions at 37 ℃ for 24 hours. Afterward, the biofilms were rinsed multiple times with sterile PBS and incubated with 2 mL of PBS, MT-MnHPs (0.3 mM), or (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (0.3 mM) for 2 hours at 37 ℃. The biofilms were then subjected to US exposure (1 MHz, 1.5 W cm⁻\u0026sup2;) for 10 minutes. Following solvent removal, 2 mL of absolute methanol was applied to fix the biofilm for 15 minutes, and the sample was subsequently stained with 2 mL of crystal violet (0.5%, v/v) for an additional 15 minutes. The residual biofilm was then rinsed with PBS and completely dissolved in 2 mL of an acetic acid solution (33%, v/v) for 10 minutes. The absorbance of the resulting solution was measured at 590 nm with a microplate reader. The residual biofilm percentage was quantified as the ratio of the absorbance of the material- or US-treated samples relative to the control absorbance. The experiments were conducted in triplicate to minimize experimental error.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro imaging of bacteria.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA purified, resuspended bacterial suspension (20 \u0026micro;L, 1.0 \u0026times; 10⁷ CFU) was incubated with MT-MnHPs (0.3 mM, 200 \u0026micro;L), (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (0.3 mM, 200 \u0026micro;L), HP (0.3 mM, 200 \u0026micro;L), maltotriose (0.3 mM, 200 \u0026micro;L), or PBS (200 \u0026micro;L) for 2 hours in a shaking incubator at 200 rpm and 37 ℃. Bacteria were then collected by centrifugation at 6000 rpm for 5 minutes in Eppendorf (EP) tubes, resuspended, and washed three times with PBS. A 10 \u0026micro;L sample of the washed bacterial suspension was then transferred onto a microscope slide, covered with a coverslip, and imaged \u003cem\u003evia\u003c/em\u003e confocal laser scanning microscopy (CLSM, Leica, TCSSP5 II) with a diode laser at 30% power. All fluorescence images were captured with CLSM under identical optical parameters, with consistent brightness and contrast applied automatically. Region-of-interest (ROI) processing and analysis were conducted \u003cem\u003evia\u003c/em\u003e Leica Application Suite Advanced Fluorescence Lite software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo imaging of bacteria.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e All procedures involving animals were approved by the Soochow University Laboratory Animal Center (SYXK(SU) 2021\u0026ndash;0073). To establish MRSA-induced nephritis, 25 \u0026micro;L of MRSA was directly injected into the kidneys of nude mice (female, 6\u0026ndash;8 weeks old; n\u0026thinsp;=\u0026thinsp;3). Twelve hours postinjection, infected mice received intravenous administration of MT-MnHPs (0.3 mM, 200 \u0026micro;L), (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (0.3 mM, 200 \u0026micro;L), HP (0.3 mM, 200 \u0026micro;L), or PBS (200 \u0026micro;L). In vivo MRI was then performed 30 minutes after injection \u003cem\u003evia\u003c/em\u003e a 3.0 T clinical MRI scanner, employing FSE T\u003csub\u003e1\u003c/sub\u003e-weighted imaging (TE\u0026thinsp;=\u0026thinsp;12 ms; TR\u0026thinsp;=\u0026thinsp;400 ms; FOV\u0026thinsp;=\u0026thinsp;80 \u0026times; 80 mm; slice thickness\u0026thinsp;=\u0026thinsp;2 mm; spacing\u0026thinsp;=\u0026thinsp;0.2 mm; matrix\u0026thinsp;=\u0026thinsp;256 \u0026times; 256) and T\u003csub\u003e2\u003c/sub\u003e-weighted imaging (TE\u0026thinsp;=\u0026thinsp;58 ms; TR\u0026thinsp;=\u0026thinsp;300 ms; FOV\u0026thinsp;=\u0026thinsp;80 \u0026times; 48 mm; slice thickness\u0026thinsp;=\u0026thinsp;2 mm; spacing\u0026thinsp;=\u0026thinsp;0.2 mm; matrix\u0026thinsp;=\u0026thinsp;256 \u0026times; 256). The local MRSA concentration at the infection site during imaging was approximately 1.0 \u0026times; 10⁸ CFU, as confirmed through kidney tissue homogenization and CFU quantification. Additionally, to assess selectivity, glycerin-induced nephritis was modelled in mice (female, 6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;3) and treated with either MT-MnHPs (0.3 mM, 200 \u0026micro;L) or Gd-DTPA (0.2 mL kg⁻\u0026sup1;, 200 \u0026micro;L) for MRI (T\u003csub\u003e1\u003c/sub\u003e-weighted: TE\u0026thinsp;=\u0026thinsp;12 ms; TR\u0026thinsp;=\u0026thinsp;400 ms; FOV\u0026thinsp;=\u0026thinsp;80 \u0026times; 80 mm; slice thickness\u0026thinsp;=\u0026thinsp;2 mm; spacing\u0026thinsp;=\u0026thinsp;0.2 mm; matrix\u0026thinsp;=\u0026thinsp;256 \u0026times; 256).\u003c/p\u003e \u003cp\u003eTo construct a tumour-containing bacterial model, we subcutaneously injected 100 \u0026micro;L of CT26 cell suspension into the right back region of nude mice (female, 6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;3). When the tumor size reached\u0026thinsp;~\u0026thinsp;100 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the mice were randomly divided into two groups. In one group, we subcutaneously injected bacteria into the left thigh (MRSA: 50 \u0026micro;L, ~\u0026thinsp;1.1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU) but not into the right tumour. In the other group, we subcutaneously injected bacteria into the left thigh (MRSA: 50 \u0026micro;L, ~\u0026thinsp;1.1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU) as well as the right tumour (MRSA: 50 \u0026micro;L, ~\u0026thinsp;1.1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU). The actual number of bacteria at the infection sites during imaging was also determined \u003cem\u003evia\u003c/em\u003e tissue harvesting, homogenization and culture with a CFU count. The final concentration of MRSA at the infection site during imaging was ~\u0026thinsp;1.0 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU. Twelve hours postinjection, infected mice received intravenous administration of MT-MnHPs (0.3 mM, 200 \u0026micro;L). In vivo MRI was then performed 4 hours after injection \u003cem\u003evia\u003c/em\u003e a 3.0 T clinical MRI scanner, employing FSE T\u003csub\u003e1\u003c/sub\u003e-weighted imaging (TE\u0026thinsp;=\u0026thinsp;17.2 ms; TR\u0026thinsp;=\u0026thinsp;629 ms; FOV\u0026thinsp;=\u0026thinsp;3 \u0026times; 3 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e; slice thickness\u0026thinsp;=\u0026thinsp;1.5 mm; spacing\u0026thinsp;=\u0026thinsp;0.2 mm; matrix\u0026thinsp;=\u0026thinsp;256 \u0026times; 256) and T\u003csub\u003e2\u003c/sub\u003e-weighted imaging (TE\u0026thinsp;=\u0026thinsp;42.1 ms; TR\u0026thinsp;=\u0026thinsp;2500 ms; FOV\u0026thinsp;=\u0026thinsp;3 \u0026times; 3 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e; slice thickness\u0026thinsp;=\u0026thinsp;1.5 mm; spacing\u0026thinsp;=\u0026thinsp;0.2 mm; matrix\u0026thinsp;=\u0026thinsp;256 \u0026times; 256).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro antibacterial assays.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe bacterial suspensions were treated under identical conditions, followed by staining with 15 \u0026micro;L of N01/PI solution (1 mg mL⁻\u0026sup1;) for 15 minutes in darkness. The stained bacteria were rinsed with sterile PBS to remove residual dye and pelleted by centrifugation (6000 rpm, 5 minutes). Viability and cell death were then visualized \u003cem\u003evia\u003c/em\u003e CLSM. Bacterial samples treated with PBS or ultrasound alone served as controls.\u003c/p\u003e \u003cp\u003eSEM was used to characterize the morphology of bacteria treated with PBS, (MT)\u003csub\u003e2\u003c/sub\u003e-MnHPs (200 \u0026micro;L, 0.3 mM), or MT-MnHPs (200 \u0026micro;L, 0.3 mM) after ultrasound exposure (1 MHz, 1.5 W cm⁻\u0026sup2;, 10 minutes). The treated bacterial suspensions were placed on silicon wafers, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and subsequently dehydrated \u003cem\u003evia\u003c/em\u003e a graded ethanol series (50%, 75%, 90%, and 100%, each for 5 minutes). The silicon wafers were dried thoroughly and coated with gold prior to SEM imaging.\u003c/p\u003e \u003cp\u003eIn a 24-well plate setup, chicken breast tissues of varying thicknesses were covered with MRSA or MDR \u003cem\u003eE. coli\u003c/em\u003e incubated with MT-MnHPs (0.3 mM). Following ultrasound treatment (1 MHz, 1.5 W cm⁻\u0026sup2; for 10 minutes), agar plate assays were conducted to quantify bacterial viability. The antibacterial rate was calculated on the basis of CFU counts on agar plates as follows:\u003c/p\u003e \u003cp\u003eAntibacterial rate (%) = (\u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003econtrol\u003c/em\u003e\u003c/sub\u003e - \u003cem\u003eN\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e)/\u003cem\u003eN\u003c/em\u003e\u003csub\u003econtrol\u003c/sub\u003e\u0026times;100% (1)\u003c/p\u003e \u003cp\u003ewhere \u0026ldquo;\u003cem\u003eN\u003c/em\u003e\u003csub\u003econtrol\u003c/sub\u003e\u0026rdquo; and \u0026ldquo;\u003cem\u003eN\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e\u0026rdquo; represent bacterial counts (CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the control groups of \u0026ldquo;PBS\u0026rdquo; and other experimental groups (experiment), respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo antibacterial assays.\u003c/b\u003e We used MRSA-induced nephritis in mice to evaluate the antibacterial ability of the developed strategy in vivo. To construct the model, MRSA (25 \u0026micro;L, ~\u0026thinsp;1.1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU) was injected in situ into the right kidney of each mouse (female, 6\u0026ndash;8 weeks old, n\u0026thinsp;=\u0026thinsp;3). At 12 h postinjection, these mice were intravenously injected with 200 \u0026micro;L of 0.3 mM MT-MnHPs or PBS buffer on days 1, 3, 5 and 7. Six hours after each drug injection, the infected sites were irradiated with or without ultrasound (1 MHz, 1.5 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10 min). After SDT, the infected kidney tissues were extracted, homogenized, and cultured on plates. The corresponding antibacterial rate was calculated \u003cem\u003evia\u003c/em\u003e Eq.\u0026nbsp;(1). After treatment, the infected kidney tissues were fixed in 4% PFA solution for H\u0026amp;E staining.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eFor statistical significance testing, we used one-way ANOVA or the paired two-tailed t test. The statistical analysis was performed \u003cem\u003evia\u003c/em\u003e Origin or GraphPad Prism software. The error bars represent the standard deviation obtained from three independent measurements. All imaging experiments were repeated three times with similar results. A region of interest (ROI) was employed for quantitative assessments of fluorescence intensity, which was calculated \u003cem\u003evia\u003c/em\u003e commercial image analysis software (Leica Application Suite Advanced Fluorescence Lite, LAS AF Lite).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLife Science Reporting Summary.\u003c/b\u003e Further information on experimental design is available in the Life Science Reporting Summary.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Ling Wen (Soochow University, China) for her general help and valuable suggestions. Y. H. discloses support for the research described in this study from the National Key R\u0026amp;D Program of China (2023YFB3208200), the National Natural Science Foundation of China [grant numbers 22393932, T2321005, 21825402], the Science and Technology Development Fund, Macau SAR [grant number 0002/2022/AKP, 0115/2023/RIA2], the Major Independent Research Project of Jiangsu Key Laboratory for Carbon-Based Functional Materials \u0026amp; Devices (grant number L421490022) and the Program for Jiangsu Specially Ap-pointed Professors to Professor Yao He, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), 111 Project and Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO-CIC). H.Y.W. provided support for the research described in this study from the National Natural Science Foundation of China [grant number 22074101].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.N.Z., Y.D.Z., J.W.Z., B.S., H.Y.W. and Y.H. conceived and designed the research. M.N.Z., Y.D.Z. and J.W.Z. carried out most of the experiments and analysed the data. P.C.W., J.L.Z., Y.Y.Z. and X.L. performed additional experiments and characterizations. M.N.Z., J.W.Z., Y.D.Z., B.S., H.Y.W. and Y.H. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e is available in the online version of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions\u003c/strong\u003e information is available online at www.nature.com/reprints.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u0026nbsp;\u003c/strong\u003eshould be addressed to B.S., H. Y. W. or Y. H.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note:\u003c/strong\u003e Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRanjbar R, Alam M (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399:629\u0026ndash;655\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkuta KS et al (2022) Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet 400:2221\u0026ndash;2248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall-Stoodley L et al (2012) Towards diagnostic guidelines for biofilm-associated infections. 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Chem Rev 99:2293\u0026ndash;2352\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5646556/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5646556/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent magnetic resonance imaging (MRI) methods often fail to differentiate bacterial infections from nonbacterial inflammatory conditions because of their poor specificity. To address this limitation, we synthesized two MRI probes that exploit bacterial-specific ATP-binding cassette (ABC) sugar transporters for the selective delivery of manganese porphyrin-based nanoparticles into antibiotic-resistant bacteria. These probes were synthesized via click chemistry by coupling azide-functionalized maltotriose with alkyne-modified manganese hematoporphyrin, which formed self-assembling nanoparticles. Our studies revealed\u0026thinsp;~\u0026thinsp;65% probe uptake in gram-positive and gram-negative bacteria, with negligible uptake (~\u0026thinsp;1%) in ABC transporter-deficient mutants. The probes demonstrated high longitudinal and transverse relaxivities (up to 11.56 mM⁻\u0026sup1;s⁻\u0026sup1; and 102.0 mM⁻\u0026sup1;s⁻\u0026sup1;, respectively), enabling ultrasensitive MRI detection of human-derived methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and multidrug-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e at concentrations as low as 10⁶ CFU. In murine models, the probes differentiated bacterial nephritis from nonbacterial inflammation and visualized bacteria within tumour tissues, outperforming clinically used gadolinium-based agents. This study provides a promising approach for precise magnetic resonance imaging of antibiotic-resistant bacterial infections in deep tissues.\u003c/p\u003e","manuscriptTitle":"Selective magnetic resonance imaging of antibiotic-resistant bacteria leveraging ATP-binding cassette sugar transporter-responsive probes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-10 18:12:11","doi":"10.21203/rs.3.rs-5646556/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a6622e6-29a6-4d2e-85d4-d99bac0ba2ac","owner":[],"postedDate":"January 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42630656,"name":"Biological sciences/Biological techniques/Imaging/Magnetic resonance imaging"},{"id":42630657,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":42630658,"name":"Physical sciences/Materials science/Biomaterials/Drug delivery"}],"tags":[],"updatedAt":"2025-02-10T16:15:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-10 18:12:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5646556","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5646556","identity":"rs-5646556","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}