Thermo-responsive cascade antimicrobial platform for precise biofilm removal and enhanced wound healing

Abstract

Background: Bacterial infection, tissue hypoxia and inflammatory response can hinder infected wound repair. This study aimed to develop a multifunctional specific therapeutic photo-activated release nanosystem [HMPB@MB@AuNPs@PMB@HA (HMAPH)] by loading photosensitizer methylene blue (MB) into hollow mesoporous Prussian blue nanostructures and modifying the surface with gold particles, polymyxin B (PMB) and hydrophilic hyaluronic acid. Methods: The HMAPH was characterized using transmission electron microscopy, UV–vis, Fourier-transform infrared spectroscopy, X-ray diffraction and X-ray photon spectroscopy. The photothermal performance, iron ion release and free radical generation of the HMAPH were measured under different conditions to investigate its thermo-responsive cascade reaction. The antibacterial ability of HMAPH was investigated using live/dead fluorescence tests. The morphology and membrane integrity of Pseudomonas aeruginosa (P. aeruginosa) were investigated using transmission electron microscopy. The anti-biofilm activity of HMAPH was evaluated using crystal violet and SYBR Green I staining. Finally, we established a mouse model of a skin wound infected by P. aeruginosa to confirm the in vivo effectiveness of HMAPH. We used immunofluorescent staining, hematoxylin–eosin staining, Masson staining and enzyme-linked immunosorbent assay to examine whether HMAPH promoted wound healing and reduced inflammatory damage. Results: In this study, hyaluronic acid was decomposed under the action of hyaluronidase. Also, the exposed nanomaterials specifically bound to the outer membrane of P. aeruginosa through PMB to increase the membrane sensitivity to photodynamic treatment. Under dual-light irradiation, a large amount of iron ions released by HMAPH underwent a Fenton reaction with H₂O₂ in bacteria to generate hydroxyl radicals (•OH), enabling direct killing of cells by hyperthermia. Additionally, the photodynamic activity of MB released by photo-induced activation led to the generation of reactive oxygen species, achieving synergistic and effective inhibition of P. aeruginosa. HMAPH also inhibited biofilm formation and downregulated the expression of virulence factors. In vivo experiments revealed that HMAPH accelerated the healing of P. aeruginosa-infected wounds by promoting angiogenesis and skin regeneration, inhibiting the inflammatory response and promoting M1 to M2 polarization. Conclusions: Our study proposed a strategy against bacteria and biofilms through a synergistic photothermal–photodynamic–Fenton reaction, opening up new prospects for combating biofilm-associated infections.

Keywords

HMAPH nanocomposites / Photothermal / Photodynamic / Fenton reaction / Synergistic antibacteria / Biofilm / Wound healing / Infection

References

[1] Xu QL, Zhu P, Zhang JR, Liu YH, Cai L, Jiang HJ, et al. Electrochemical formation of distinct nanostructured MoS2 with altered antibacterial activity. Mater Lett 2020; 271: 127809. https://doi.org/10.1016/j.matlet.2020.127809.

[2] Cai L, Huang YQ, Duan YY, Liu Q, Xu Q, Jia J, et al. Schiff-base silver nanocomplexes formation on natural biopolymer coated mesoporous silica contributed to the improved curative effect on infectious microbes. Nano Res 2021; 14: 2735-48. https://doi.org/10.1007/s12274-020-3279-6.

[3] Qian X, Lu TL, Huang CQ, Zheng DW, Gong GC, Chu X, et al. Bioinspired sonodynamic nano spray accelerates infected wound healing via targeting and disturbing bacterial metabolism. Adv Funct Mater 2024; 34: 2315576. https://doi.org/10.1002/adfm.202315576.

[4] Cai L, Zhu XY, Ruan HJ, Yang J, Wei W, Wu Y, et al. Curcumin-stabilized silver nanoparticles encapsulated in biocompatible electrospun nanofibrous scaffold for sustained eradication of drug-resistant bacteria. J Hazard Mater 2023; 452: 131290. https://doi.org/10.1016/j.jhazmat.2023.131290.

[5] Zhu P, Zhou LZ, Song YY, Cai L, Ji MH, Wang J, et al. Encapsulating insoluble antifungal drugs into oleic acid-modified silica mesocomposites with enhanced fungicidal activity. J Mater Chem B 2020; 8: 4899-907. https://doi.org/10.1039/D0TB00106F.

[6] Xu QL, Zhang L, Liu YH, Cai L, Zhou LZ, Jiang H, et al. Encapsulating MoS2-nanoflowers conjugated with chitosan oligosaccharide into electrospun nanofibrous scaffolds for photothermal inactivation of bacteria. J Nanostructure Chem 2024; 14: 137-51. https://doi.org/10.1007/s40097-022-00494-1.

[7] Zhu XY, Wang J, Cai L, Wu Y, Ji MH, Jiang HJ, et al. Dissection of the antibacterial mechanism of zinc oxide nanoparticles with manipulable nanoscale morphologies. J Hazard Mater 2022; 430: 128436. https://doi.org/10.1016/j.jhazmat.2022.128436.

[8] Sommer R, Wagner S, Rox K, Varrot A, Hauck D, Wamhoff EC, et al. Glycomimetic, orally bioavailable lecb inhibitors block biofilm formation of Pseudomonas aeruginosa. J Am Chem Soc 2018; 140: 2537-45. https://doi.org/10.1021/jacs.7b11133.

[9] Chen MH, Wei JJ, Xie SZ, Tao XY, Zhang ZL, Ran P, et al. Bacterial biofilm destruction by size/surface charge-adaptive micelles. Nanoscale 2019; 11: 1410-22. https://doi.org/10.1039/C8NR05575K.

[10] Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000; 406: 959-64. https://doi.org/10.1038/35023079.

[11] Li J, Zhang K, Ruan L, Chin SF, Wickramasinghe N, Liu H, et al. Block copolymer nanoparticles remove biofilms of drug-resistant gram-positive bacteria by nanoscale bacterial debridement. Nano Lett 2018; 18: 4180-7. https://doi.org/10.1021/acs.nanolett.8b01000.

[12] Durmus NG, Taylor EN, Kummer KM, Webster TJ. Enhanced efficacy of superparamagnetic iron oxide nanoparticles against antibiotic-resistant biofilms in the presence of metabolites. Adv Mater 2013; 25: 5706-13. https://doi.org/10.1002/adma.201302627.

[13] Flemming HC, Wuertz S. Bacteria and archaea on earth and their abundance in biofilms. Nat Rev Microbiol 2019; 17: 247-60. https://doi.org/10.1038/s41579-019-0158-9.

[14] Wagner S, Sommer R, Hinsberger S, Lu C, Hartmann RW, Empting M, et al. Novel strategies for the treatment of pseudomonas aeruginosa infections. J Med Chem 2016; 59: 5929-69. https://doi.org/10.1021/acs.jmedchem.5b01698.

[15] Qin SY, Zhang AQ, Zhang XZ. Recent advances in targeted tumor chemotherapy based on smart nanomedicines. Small 2018; 14: e1802417. https://doi.org/10.1002/smll.201802417.

[16] Riley RS, June CH, Langer R, Mitchell MJ. Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov 2019; 18: 175-96. https://doi.org/10.1038/s41573-018-0006-z.

[17] Zhang XG, Tang JJ, Li C, Lu Y, Cheng LL, Liu J. A targeting black phosphorus nanoparticle based immune cells nano-regulator for photodynamic/photothermal and photoimmunotherapy. Bioact Mater 2021; 6: 472-89. https://doi.org/10.1016/j.bioactmat.2020.08.024.

[18] Yu Z, Chan WK, Zhang Y, Tan TY. Near-infrared-II activated inorganic photothermal nanomedicines. Biomaterials 2021; 269: 120459. https://doi.org/10.1016/j.biomaterials.2020.120459.

[19] Lian H, Guan P, Tan H, Zhang X, Meng Z. Near-infrared light triggered multi-hit therapeutic nanosystem for tumor specific photothermal effect amplified signal pathway regulation and ferroptosis. Bioact Mater 2022; 9: 63-76. https://doi.org/10.1016/j.bioactmat.2021.07.014.

[20] Lan M, Zhao S, Liu W, Lee CS, Zhang W, Wang P. Photosensitizers for photodynamic therapy. Adv Healthc Mater 2019; 8: 1900132. https://doi.org/10.1002/adhm.201900132.

[21] Luo Y, Li J, Liu X, Tan L, Cui Z, Feng X, et al. Dual metal-organic framework heterointerface. ACS Cent Sci 2019; 5: 1591-601. https://doi.org/10.1021/acscentsci.9b00639.

[22] Wei S, Qiao Y, Wu Z, Liu X, Li Y, Cui Z, et al. Na⁺ inserted metal-organic framework for rapid therapy of bacteria-infected osteomyelitis through microwave strengthened Fenton reaction and thermal effects. Nano Today 2021; 37: 101090. https://doi.org/10.1016/j.nantod.2021.101090.

[23] Yuan Z, Lin C, He Y, Tao B, Chen M, Zhang J, et al. Near-infrared light-triggered nitric-oxide-enhanced photodynamic therapy and low-temperature photothermal therapy for biofilm elimination. ACS Nano 2020; 14: 3546-62. https://doi.org/10.1021/acsnano.9b09871.

[24] Wang Y, Xu Y, Guo X, Wang L, Zeng J, Qiu H, et al. Enhanced antimicrobial activity through the combination of antimicrobial photodynamic therapy and low-frequency ultrasonic irradiation. Adv Drug Deliv Rev 2022; 183: 114168. https://doi.org/10.1016/j.addr.2022.114168.

[25] Han X, Boix G, Balcerzak M, Moriones OH, Sarabia M, Cortés P, et al. Antibacterial films based on MOF composites that release iodine passively or upon triggering by near-infrared light. Adv Funct Mater 2022; 32: 2112902. https://doi.org/10.1002/adfm.202112902.

[26] Xu M, Ji X, Huo J, Chen J, Liu N, Li Z, et al. Nonreleasing AgNP colloids composite hydrogel with potent hemostatic, photodynamic bactericidal and wound healing-promoting properties. ACS Appl Mater Interfaces 2023; 15: 17742-56. https://doi.org/10.1021/acsami.3c03247.

[27] Tang Q, Xiao W, Huang C, Si W, Shao J, Huang W, et al. pH-triggered and enhanced simultaneous photodynamic and photothermal therapy guided by photoacoustic and photothermal imaging. Chem Mater 2017; 29: 5216-24. https://doi.org/10.1021/acs.chemmater.7b01075.

[28] Yuan H, Hong X, Ma H, Fu C, Guan Y, Huang W, et al. MXene-based dual functional nanocomposite with photothermal nanozyme catalytic activity to fight bacterial infections. ACS Mater Lett 2023; 5: 762-74. https://doi.org/10.1021/acsmaterialslett.2c00771.

[29] Wang Y, Jin Y, Chen W, Wang J, Chen H, Sun L, et al. Construction of nanomaterials with targeting phototherapy properties to inhibit resistant bacteria and biofilm infections. Chem Eng J 2019; 358: 74-90. https://doi.org/10.1016/j.cej.2018.10.002.

[30] Liu B, Li CX, Chen GY, Liu B, Deng XR, Wei Y, et al. Synthesis and optimization of MoS2@Fe3O4-ICG/Pt (IV) nanoflowers for MR/IR/PA bioimaging and combined PTT/PDT/chemotherapy triggered by 808 nm laser. Adv Sci 2017; 4: 1600540.

[31] Younis MR, Wang C, An R, Wang S, Younis MA, Li ZQ, et al. Low power single laser activated synergistic cancer phototherapy using photosensitizer functionalized dual plasmonic photothermal nanoagents. ACS Nano 2019; 13: 2544-57. https://doi.org/10.1021/acsnano.8b09552.

[32] Liu Y, Guo Z, Li F, Xiao Y, Zhang Y, Bu T, et al. Multifunctional magnetic copper ferrite nanoparticles as Fenton-like reaction and near-infrared photothermal agents for synergetic antibacterial therapy. ACS Appl Mater Interfaces 2019; 11: 31649-60. https://doi.org/10.1021/acsami.9b10096.

[33] Zhang W, Hu S, Yin JJ, He W, Lu W, Ma M, et al. Prussian blue nanoparticles as multienzyme mimetics and reactive oxygen species scavengers. J Am Chem Soc 2016; 138: 5860-5. https://doi.org/10.1021/jacs.5b12070.

[34] Li J, Liu X, Tan L, Cui Z, Yang X, Liang Y, et al. Zinc-doped Prussian blue enhances photothermal clearance of Staphylococcus aureus and promotes tissue repair in infected wounds. Nat Commun 2019; 10: 4490. https://doi.org/10.1038/s41467-019-12429-6.

[35] Andrews SC, Robinson AK, Quiñones F. Bacterial iron homeostasis. FEMS Microbiol Rev 2003; 27: 215-37. https://doi.org/10.1016/S0168-6445(03)00055-X.

[36] Hu R, Fang Y, Huo M, Yao H, Wang C, Chen Y, et al. Ultrasmall Cu2-xS nanodots as photothermal-enhanced Fenton nanocatalysts for synergistic tumor therapy at NIR-II biowindow. Biomaterials 2019; 206: 101-14. https://doi.org/10.1016/j.biomaterials.2019.03.014.

[37] Xie X, Wang R, Zhang X, Ren Y, Du T, Ni Y, et al. A photothermal and self-induced Fenton dual-modal antibacterial platform for synergistic enhanced bacterial elimination. Appl Catal B Environ 2021; 295: 120315. https://doi.org/10.1016/j.apcatb.2021.120315.

[38] Zhou F, Lin S, Zhang J, Kong Z, Tan BK, Hamzah SS, et al. Enhancement of photodynamic bactericidal activity of curcumin against Pseudomonas aeruginosa using polymyxin B. Photodiagn Photodyn Ther 2022; 37: 102677. https://doi.org/10.1016/j.pdpdt.2021.102677.

[39] Wu W, Yu L, Pu Y, Yao H, Chen Y, Shi J. Copper-enriched prussian blue nanomedicine for in situ disulfiram toxification and photothermal antitumor amplification. Adv Mater 2020; 32: e2000542. https://doi.org/10.1002/adma.202000542.

[40] Su CH, Li WP, Tsao LC, Wang LC, Hsu YP, Wang WJ, et al. Enhancing microcirculation on multitriggering manner facilitates angiogenesis and collagen deposition on wound healing by photoreleased NO from hemin-derivatized colloids. ACS Nano 2019; 13: 4290-301. https://doi.org/10.1021/acsnano.8b09417.

[41] Ren B, Jones LA, Oppedisano DK, Kandjani AE, Chen M, Antolasik F, et al. The preparation of a AuCN/Prussian blue nanocube composite through galvanic replacement enhances stability for electrocatalysis. Chemistry Select 2017; 2: 5333-40. https://doi.org/10.1002/slct.201700908.

[42] Wang C, Wang Y, Xu L, Zhang D, Liu M, Li X, et al. Facile aqueous-phase synthesis of biocompatible and fluorescent Ag2S nanoclusters for bioimaging: tunable photoluminescence from red to near infrared. Small 2012; 8: 3137-42. https://doi.org/10.1002/smll.201200376.

[43] Wang Y, Zhao Y, Wu J, Li M, Tan J, Fu W, et al. Negatively charged sulfur quantum dots for treatment of drug-resistant pathogenic bacterial infections. Nano Lett 2021; 21: 9433-41. https://doi.org/10.1021/acs.nanolett.1c02697.

[44] Li J, Zhang Q, Chen Z, Guo S, Guo J, Yan F. Postsynthetic modification of thermo-treated metal-organic framework for combined photothermal/photodynamic antibacterial therapy. ACS Appl Mater Interfaces 2024; 16: 8459-73. https://doi.org/10.1021/acsami.3c17955.

[45] Ci D, Wang N, Xu Y, Wu S, Wang J, Li H, et al. SiO2@AuAg/PDA hybrid nanospheres with photo-thermally enhanced synergistic antibacterial and catalytic activity. RSC Adv 2024; 14: 4518-32. https://doi.org/10.1039/D3RA07607E.

[46] Wang C, Chen Q, Yin R, Yuan X, Kang H, Cai A, et al. Photothermal effect and antimicrobial properties of cerium-doped bioactive glasses. Ceram Int 2024; 50: 20235-46. https://doi.org/10.1016/j.ceramint.2024.03.147.

[47] Xie M, Gao M, Yun Y, Malmsten M, Rotello VM, Zboril R, et al. Antibacterial nanomaterials: mechanisms, impacts on antimicrobial resistance and design principles. Angew Chem Int Ed 2023; 62:e202217345. https://doi.org/10.1002/anie.202217345.

[48] Miller MB, Bassler BL. Quorum sensing in bacteria. Ann Rev Microbiol 2001; 55: 165-99. https://doi.org/10.1146/annurev.micro.55.1.165.

[49] Welsh MA, Eibergen NR, Moore JD, Blackwell HE. Small molecule disruption of quorum sensing cross-regulation in Pseudomonas aeruginosa causes major and unexpected alterations to virulence phenotypes. J Am Chem Soc 2015; 137: 1510-9. https://doi.org/10.1021/ja5110798.

[50] Kong KF, Jayawardena SR, Indulkar SD, del Puerto A, Koh CL, Høiby N, et al. Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB β-lactamases, proteases, quorum sensing, and other virulence factors. Antimicrob Agents Chemother 2005; 49: 4567-75. https://doi.org/10.1128/AAC.49.11.4567-4575.2005.

[51] Li Z, You S, Mao R, Xiang Y, Cai E, Deng H, et al. Architecting polyelectrolyte hydrogels with Cu-assisted polydopamine nanoparticles for photothermal antibacterial therapy. Mater Today Bio 2022; 15: 100264. https://doi.org/10.1016/j.mtbio.2022.100264.

[52] Yin M, Wu J, Deng M, Wang P, Ji G, Wang M, et al. Multifunctional magnesium organic framework-based microneedle patch for accelerating diabetic wound healing. ACS Nano 2021; 15: 17842-53. https://doi.org/10.1021/acsnano.1c06036.

[53] He X, Dai L, Ye L, Sun X, Enoch O, Hu R, et al. A vehicle-free antimicrobial polymer hybrid gold nanoparticle as synergistically therapeutic platforms for Staphylococcus aureus infected wound healing. Adv Sci 2022; 9: 2105223. https://doi.org/10.1002/advs.202105223.

[54] Deng Z, Shi F, Zhou Z, Sun F, Sun MH, Sun Q, et al. M1 macrophage mediated increased reactive oxygen species (ROS) influence wound healing via the MAPK signaling in vitro and in vivo. Toxicol Appl Pharmacol 2019; 366: 83-95. https://doi.org/10.1016/j.taap.2019.01.022.

[55] Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 2011; 11: 723-37. https://doi.org/10.1038/nri3073.

[56] Hernando S, Coque TM, Baquero F, Martínez JL. Defining and combating antibiotic resistance from one health and global health perspectives. Nat Microbiol 2019; 4: 1432-42. https://doi.org/10.1038/s41564-019-0503-9.

[57] Reyes J, Komarow L, Chen L, Ge L, Hanson BM, Cober E, et al. Global epidemiology and clinical outcomes of carbapenem-resistant Pseudomonas aeruginosa and associated carbapenemases (POP): a prospective cohort study. Lancet Microbe 2023; 4: e159-70. https://doi.org/10.1016/S2666-5247(22)00329-9.

[58] Huang T, He X, Ali A, Gnanasekar S, Xiang Y, Zhang K, et al. Phytic acid-promoted deposition of gold nanoparticles with grafted cationic polymer brushes for the construction of synergistic contact-killing and photothermal bactericidal coatings. ACS Appl Bio Mater 2024; 7: 3283-94. https://doi.org/10.1021/acsabm.4c00237.

[59] Tian H, Zhu H, Xue Y, Wang M, Xing K, Li Z, et al. White light powered antimicrobial nanoagents for triple photothermal, chemodynamic and photodynamic based sterilization. Nanoscale Horiz 2024; 9: 659-874. https://doi.org/10.1039/D4NH00060A.

[60] Li J, Pan G, Zyryanov GV, Peng Y, Zhang G, Ma L, et al. Positively charged semiconductor conjugated polymer nanomaterials with photothermal activity for antibacterial and antibiofilm activities in vitro and in vivo. ACS Appl Mater Interfaces 2023; 15: 40864-76. https://doi.org/10.1021/acsami.3c00556.

[61] Zhong J, Xiao F, Jin B, Zhang G, Hou H, Bi J, et al. Construction of sandwich-structured V2CTx@PDA@Ag nanosheet composite for enhanced near-infrared photothermocatalytic sterilization performance. Mater Chem Phys 2024; 314: 128931. https://doi.org/10.1016/j.matchemphys.2024.128931.

[62] Xu B, Yu D, Xu C, Gao Y, Sun H, Liu L, et al. Study on synergistic mechanism of molybdenum disulfide/sodium carboxymethyl cellulose composite nanofiber mats for photothermal/photodynamic antibacterial treatment. Int J Biol Macromol 2024; 266: 130838. https://doi.org/10.1016/j.ijbiomac.2024.130838.

[63] Li X, Zhou J, Dong X, Cheng WY, Duan H, Cheung PCK. In Vitro and In Vivo Photothermal cancer therapeutic effects of gold nanorods modified with mushroom β-Glucan. J Agric Food Chem 2018; 66: 4091-8. https://doi.org/10.1021/acs.jafc.8b00292.