Cholesterol-Assisted Bacterial Cell Surface Engineering for Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

Abstract

Antibacterial photodynamic therapy (PDT), which enables effective killing of regular and multidrug-resistant (MDR) bacteria, is a promising treatment modality for bacterial infection. However, because most photosensitizer (PS) molecules fail to strongly interact with the surface of Gram-negative bacteria, this technique is suitable for treating only Gram-positive bacterial infection, which largely hampers its practical applications. Herein, we reveal for the first time that cholesterol could significantly facilitate the hydrophobic binding of PSs to the bacterial surface, achieving the hydrophobic interaction-based bacterial cell surface engineering that could effectively photoinactivate both Gram-negative and Gram-positive bacteria. An amphiphilic polymer composed of a polyethylene glycol (PEG) segment terminated with protoporphyrin IX (PpIX, an anionic PS) and cholesterol was constructed (abbreviated Chol-PEG-PpIX), which could self-assemble into micelle-like nanoparticles (NPs) in aqueous solution. When encountering the Gram-negative Escherichia coli cells, the Chol-PEG-PpIX NPs would disassemble and the PpIX moieties could effectively bind to the bacterial surface with the help of the cholesterol moieties, resulting in the significantly enhanced fluorescence emission of the bacterial surface. Under white light irradiation, the light-triggered singlet oxygen (O) generation of the membrane-bound PpIX could not only severely damage the outer membrane but also facilitate the entry of external Chol-PEG-PpIX into the bacteria, achieving >99.99% bactericidal efficiency. Besides, as expected, the Chol-PEG-PpIX NPs also exhibited excellent antibacterial performance against the Gram-positive Staphylococcus aureus. We also verified that this nanoagent possesses negligible dark cytotoxicity toward mammalian cells and good hemocompatibility. To the best of our knowledge, this study demonstrates for the first time the feasibility of constructing a fully hydrophobic interaction-based and outer membrane-anchored antibacterial PDT nanoagent.

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“…Then, white light (400–750 nm) from a light emitting diode lamp with a power density of 10 mW cm −2 was used as the visible light irradiation source. [ 15 ] During the light irradiation, the fluorescence spectra of TA were monitored at an interval of 5 min for 30 min. The 426 nm emission intensity of TAOH was plotted as a function of the irradiation time.…”

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“…However, dark toxicity was usually observed when long alkyl chains were incorporated into the structures, due to polarization and rupture of bacterial cell membranes in the dark . Recently, based on hydrophobic interaction, an outer‐membrane‐anchored aPDT nanoagent was synthesized by conjugation of protoporphyrin IX and cholesterol . This nanoagent enabled effective binding to the outer membrane of Gram‐negative E. coli , resulting in a PS‐enriched bacterial surface.…”

“…[22] Recently,b ased on hydrophobic interaction, an outer-membraneanchored aPDT nanoagent was synthesized by conjugation of protoporphyrin IX and cholesterol. [23] This nanoagent enabled effective binding to the outer membrane of Gram-negative E. coli,r esulting in aP S-enriched bacterials urface. Taking advantage of the membrane accumulating ability of aromatics ubstituents, Bazan et al preparedamembrane-intercalating conjugated oligo electrolyte that showed high antibacterial ability upon irradiation.…”

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“…The characteristic red emission was seen from the cells, as previously observed in different microorganisms. [48,49] Thus, this emission can be assigned to TPCF 20 -NMe 2 bound to E. coli cells, whichremains off at neutral pH and is turned on at acidic pH.…”