Title: Breaking Iron Homeostasis: Iron Capturing Nanocomposites for Combating Bacterial Biofilm Bacterial Resistance Reversal and Inflammation Relief

Journal: Angewandte Chemie International Edition

IF: 16.1

DOI: https://doi.org/10.1002/anie.202319690

Reporter:Yajie Wang Master's Student,Class of 2023

Given the scarcity of novel antibiotics, the eradication of bacterial biofilm infections poses formidable challenges. Upon bacterial infection, the host restricts Fe ions, which are crucial for bacterial growth and maintenance. Having coevolved with the host, bacteria developed adaptive pathways like the hemin-uptake system to avoid iron deficiency. Inspired by this, we propose a novel strategy, termed iron nutritional immunity therapy (INIT), utilizing Ga-CT@P nanocomposites constructed with gallium, copper-doped tetrakis (4-carboxyphenyl) porphyrin (TCPP) metal–organic framework, and polyamine-amine polymer dots, to target bacterial iron intakes and starve them. Owing to the similarity between iron/hemin and gallium/TCPP, gallium-incorporated porphyrin potentially deceives bacteria into uptaking gallium ions and concurrently extracts iron ions from the surrounding bacteria milieu through the porphyrin ring. This strategy orchestrates a “give and take” approach for Ga³⁺/Fe³⁺ exchange. Simultaneously, polymer dots can impede bacterial iron metabolism and serve as real-time fluorescent iron-sensing probes to continuously monitor dynamic iron restriction status. INIT based on Ga-CT@P nanocomposites induced long-term iron starvation, which affected iron-sulfur cluster biogenesis and carbohydrate metabolism, ultimately facilitating biofilm eradication and tissue regeneration. Therefore, this study presents an innovative antibacterial strategy from a nutritional perspective that sheds light on refractory bacterial infection treatment and its future clinical application.

Bacterial infection is currently regarded as one of the most challenging issues, and it is the leading cause of clinical infectious deaths. Bacteria can spend years in a dormant, biochemically inactive state, but they can be revived in certain nutrient-rich environments. Conventional antibacterial therapies depend predominantly on antibiotics, which do not keep pace with the emergence of multidrug-tolerant pathogens. Eventually, a deficit in antibiotic resistance can lead to infection-related deaths. Therefore, it is imperative to develop novel therapeutic approaches to combat pathogenic biofilms during wound infections.

Trace metals are fundamental micronutrients required for the survival of all life forms. During bacterial infection, one of the host defense mechanisms involves the sequestration of essential metal ions, specifically Fe, resulting in a phenomenon known as nutritional immunity. Having coevolved with the host, pathogens have developed adaptive pathways to avoid iron starvation, for example, by stealing iron from the host through specific iron chelators called siderophores with high binding affinity. Despite the host's iron-restricted environment, sufficient siderophores and evolved iron uptake systems can satisfy the iron requirements of the pathogens. Iron competition occurs during all stages of infection, and it is challenging for the hosts to control fast-growing pathogens in the long term. Therefore, the development of a novel strategy to cope with the evolution of iron-seizing bacteria is essential for antibacterial nutritional immunity therapy.

Inspired by this nutritional immunity strategy, a “give-and-take” strategy is proposed. This approach involves the simultaneous removal of Fe³⁺ from the extracellular milieu and the introduction of fake Fe³⁺ to deceive bacterial ingestion mechanisms. This pioneering iron nutritional immunity therapy (INIT) achieves a dual iron nutritional immunity effect, potentially starving the pathogens to death. Gallium (Ga³⁺) is a semi-metallic element that resembles Fe³⁺ in both ionic radius and chemical characteristics. Microorganisms face challenges in distinguishing between Ga³⁺ and Fe³⁺. Unlike Fe³⁺, Ga³⁺ is redox-inactive under physiological conditions and cannot be directly involved in electron exchange reactions, thus suggesting potential for application in INIT. However, because of the hydrolysis of Ga³⁺, the use of Ga³⁺ alone, even at high concentrations, is only effective against a subset of bacterial strains. Therefore, selecting a carrier is crucial for our approach; hence, we considered structural resemblance to hemin. The pivotal role of hemin in the body, particularly its ability to sequester iron ions into the center of its porphyrin ring, prompted us to explore a carrier that could mimic this functionality. In this context, tetrakis (4-carboxyphenyl) porphyrin (TCPP) has emerged as a promising candidate because of its structural similarity to hemin. The structural similarity between TCPP and hemin provides the rationale for their comparable functionality, making TCPP a suitable carrier for ion exchange.

Furthermore, TCPP and hemin molecules both share a porphyrin ring comprising four pyrrole subunits linked by methine bridges. Hemin sequesters Fe³⁺ ions into the center of the porphyrin ring and functions as an iron transporter in the host. Considering the high similarity between TCPP and hemin, Ga³⁺-incorporated TCPP could synergistically enhance the INIT because the vacancies in the porphyrin ring have a stronger binding effect with Fe³⁺ than with Ga³⁺. Upon contact with bacteria, Ga³⁺-incorporated TCPP can efficiently release Ga³⁺ ions via an innovative structural framework while sequestering Fe³⁺ ions from the bacterial surroundings. In this case, the “give-and-take” strategy could synergistically induce bacterial iron starvation until death. Additionally, TCPP has been demonstrated to be a promising template for preparing photo-responsive two-dimensional metal–organic frameworks (MOFs) that exhibit excellent photothermal and photodynamic properties. herefore, TCPP-based two-dimensional MOFs can accelerate photo-triggered ion release, potentially achieving efficient Ga³⁺/Fe³⁺ ion exchange.

In this study, we utilized Ga³⁺-incorporated TCPP as a photosensitizer to coordinate Cu²⁺ to obtain Ga/Cu-TCPP (Ga-CT) nanocomposites (NCs) that facilitate irradiation-responsive photothermal therapy (PTT) and photodynamic therapy (PDT). A polyamine-amine polymer dot (PAMAM PDs) probe was introduced to enable localization and real-time monitoring, as well as to improve solubility and construct Ga-CT@PAMAM PDs (Ga-CT@P) NCs. The photothermal properties of Ga-CT@P promoted the molecular thermal motion of Ga³⁺, further accelerating the Ga³⁺/Fe³⁺ exchange in the porphyrin ring (giving Ga³⁺ to bacteria and taking Fe³⁺ from their surroundings) within the bacterial environment. Notably, Ga-CT@P NCs synergistically achieve a “three-in-one” PDT/ PTT/INIT therapy. Consequently, with our novel Ga-CT@P NCs developed based on the INIT strategy, mild heat-induced iron starvation resulted in iron-sulfur cluster metabolism disorders, glycolysis, and carbohydrate dysregulation, ultimately leading to bacterial death. The INIT validated the first definitive mechanism of iron-blocking therapy against bacterial infections. Through a comprehensive analysis of these dynamics, iron nutritional immunity therapy can shed light on the potential of Ga³⁺-mediated strategies to perturb bacterial iron homeostasis, thereby providing promising prospects for the treatment of refractory bacterial infection and future clinical applications in wound healing (Scheme 1).

1、 Synthesis and Characterization of Ga-CT and Ga-CT@P

Figure 1. Characterization of CT, Ga-CT, and Ga-CT@P nanocomposites: a) synthesis of Ga-CT@P; b) TEM image of Ga-CT; c) TEM image of Ga-CT@P; d) HRTEM image of Ga-CT@P; e) AFM height images of the prepared Ga-CT; f) elemental mapping of Ga-CT@P; g) UV/Vis absorption spectra of Ga-CT, CT, and Ga-CT@P; h) N1s XPS spectrum of Ga-CT; i) HOMO–LUMO gap of Cu-TCPP and Ga-TCPP; j) Fourier-transform infrared spectroscopy (FTIR) spectra of TCPP, PAMAM PDs, and Ga-CT@P; k) zeta potential of CT, Ga-CT, PAMAM PDs, and Ga-CT@P; and l)Fluorescence emission spectra of PAMAM PDs and Ga-CT@P (inset: Fluorescence imaging under UV light excitation. From left to right: 1:PAMAM PDs, 2: PBS, 3: Ga-CT@P). (CT: Cu-TCPP, Ga-CT: Ga/Cu-TCPP, Ga-CT@P: Ga/Cu-TCPP@PAMAM PDs).

2、In vitro Photothermal, Photodynamic, and Luminous Activity

Figure 2. Assessment of photothermal, photodynamic and Fe³⁺ sensing effects: a) schematic illustration of the photothermal and photodynamic activities of Ga-CT@P; b) infrared thermal images of numerous nanocomposites (500 μg mL⁻¹) under an 808 nm laser irradiation (1.0 W cm⁻²) for 10 min; c) photothermal heating curves of CT, Ga-CT, and Ga-CT@P under an 808 nm laser irradiation (1.0 W cm⁻²); d) photothermal cycle profiles irradiated by four on/off cycles under a laser irradiation of 808 nm (1.0 W cm⁻²); e) time-dependent absorbance spectra of CT, Ga-CT, and Ga-CT@P relative to 9, 10-anthracenediylbis (methylene) dimalonic acid (ABDA) under a 660 nm laser irradiation (1.0 W cm⁻²); f) time-dependent absorbance spectra of Ga-CT@P (500 μg mL⁻¹) reacting with ABDA under a 660 nm laser irradiation (1.0 W cm⁻²); g) metal ion selectivity results from fluorescence intensity curves; h) fluorescence intensity curves of Fe³⁺ detection by PAMAM PDs at Fe³⁺ concentrations from 10 μM to 150 μM (the small picture shows the fluorescence image under UV light excitation); i) linear relationship between fluorescence intensity and Fe³⁺ concentration; j) Schematic diagram of Ga³⁺/Fe³⁺ exchange strategy (OMR: outer membrane receptor); and k) electrostatic potential energies of Ga³⁺- and Fe³⁺-incorporated TCPP structures. (CT: Cu-TCPP, Ga-CT: Ga/Cu-TCPP, and Ga-CT@P: Ga/Cu-TCPP@PAMAM PDs).

3、In vitro Antibacterial Activity Assay

Figure 3. Antibiofilm properties of Ga-CT@P NCs: a) images of E. coli and S. aureus biofilm colonies on agar plates after different treatments; b) corresponding statistical data of per milliliter CFU colonies of E. coli and S. aureus biofilm; c) representative 3D reconstructions of the bacterial (live/dead (Syto9/PI) staining of S. aureus and E. coli biofilm); d) representative SEM images of E. coli and S. aureus biofilm; e) E. coli and S. aureus biofilm colonies on chrome azurol sulphonate agar plates after different treatments; f) corresponding statistical data of the siderophore production of E. coli and S. aureus biofilm; g) Annexin V-FITC/PI of E. coli biofilm after incubation with various groups. (CT: Cu-TCPP, Ga-CT: Ga/Cu-TCPP, and Ga-CT@P: Ga/Cu-TCPP@PAMAM PDs)

4、Density Function Theory Calculation of Ga3+-Enterobactin

Figure 4. Summary of the DFT calculation results: a) Calculated bond length, binding energies, and Eb/atom of Fe³⁺- and Ga³⁺-enterobactin; b) calculated frontier molecular orbital energy level diagrams for Fe³⁺- and Ga³⁺-enterobactin; and c) schematic diagram of the iron nutritional immunity mechanism of Ga-CT@P nanocomposites.

5. Ga-CT@P Target Iron Homeostasis through INIT

Figure 5. RNA-seq of E. coli treated with Ga-CT@P. a) Volcano map for the distribution of DEGs (gray: genes that are not significantly changed; blue: down-regulated genes; and red: up-regulated genes); b) network analysis of DEGs in the Ga-CT@P-treated strain group; GO enrichment analysis of c) downregulated and d) upregulated DEGs; KEGG enrichment of e) downregulated and f) upregulated genes; g) heat map of the DEGs associated with iron metabolism; h) heat map of the DEGs associated with carbohydrate metabolism (red: relatively high expressed genes and blue: relatively low expressed genes); i) schematic mechanism of Ga-CT@P-induced iron nutritional immunity therapy. (Ga-CT@P: Ga/Cu-TCPP@PAMAM PDs, OM: outer membrane, IM: inner membrane, and OMR: outer membrane receptor).

6、In vivo Antibacterial Activity Assay

Figure 6. Ga-CT@P NCs eliminating wound infection in vivo. a) In vivo antibacterial study procedures; b) macroscopic photos of skin wounds and relative wound healing areas in various treatment groups; c) representative changes in wound size over time; d) images of E. coli colonies on agar plates; e) corresponding statistical data; f) representative H&E and Masson's trichrome staining images; g) corresponding percentage of neutrophils; h) corresponding collagen deposition percentage; i) representative immunofluorescence images of IL-1β and Arg-1 after different treatments; and corresponding quantitative data of the percentage of j) IL-1β and k) Arg-1. n=6. (CT: Cu-TCPP, Ga-CT: Ga/Cu-TCPP, and Ga-CT@P: Ga/Cu-TCPP@PAMAM PDs.).

Conclusion:

In this study, we successfully synthesized novel Ga-CT@P NCs, with a 2-log CFU reduction, for antibacterial infection and biofilm formation. These results verify that Ga-CT@P NCs successfully combat biofilm infection and target bacterial iron nutritional metabolism through synergistic photothermal/photodynamic/iron nutritional immunity therapy. Firstly, the Ga-CT@P NCs utilize positively charged PAMAM PDs to capture bacteria on a negatively charged surface. They possess significant synergistic photothermal and photodynamic capacities. Next, Ga³⁺-incorporated porphyrin, which releases Ga³⁺ ions upon irradiation, deceives bacteria into uptaking and concurrently extracts Fe³⁺ ions from the surrounding bacteria milieu through the porphyrin ring. This strategy imposes an iron-deficient environment on the bacteria and starving them to death, which is referred to as iron nutritional immunity therapy. Simultaneously, the PAMAM PDs in the designed Ga-CT@P NCs function as monitors observing the dynamic iron-restriction in bacteria. Furthermore, the antibacterial mechanism was investigated at the transcriptomic level. The INIT strategy contributes to the disruption of iron-sulfur cluster metabolism, TCA cycle, and ATP metabolism. Overall, the INIT strategy embodied by Ga-CT@P provides a new perspective for the future treatment of bacterial infections by disrupting bacterial nutritional homeostasis.