Oxidization enhances type I ROS generation of AIE-active zwitterionic photosensitizers for photodynamic killing of drug-resistant bacteria

Type I photosensitizers (PSs) with an aggregation-induced emission (AIE) feature have received sustained attention for their excellent theranostic performance in the treatment of clinical diseases. However, the development of AIE-active type I PSs with strong reactive oxygen species (ROS) production capacity remains a challenge due to the lack of in-depth theoretical studies on the aggregate behavior of PSs and rational design strategies. Herein, we proposed a facile oxidization strategy to enhance the ROS generation efficiency of AIE-active type I PSs. Two AIE luminogens, MPD and its oxidized product MPD-O were synthesized. Compared with MPD, the zwitterionic MPD-O showed higher ROS generation efficiency. The introduction of electron-withdrawing oxygen atoms results in the formation of intermolecular hydrogen bonds in the molecular stacking of MPD-O, which endowed MPD-O with more tightly packed arrangement in the aggregate state. Theoretical calculations demonstrated that more accessible intersystem crossing (ISC) channels and larger spin–orbit coupling (SOC) constants provide further explanation for the superior ROS generation efficiency of MPD-O, which evidenced the effectiveness of enhancing the ROS production ability by the oxidization strategy. Moreover, DAPD-O, a cationic derivative of MPD-O, was further synthesized to improve the antibacterial activity of MPD-O, showing excellent photodynamic antibacterial performance against methicillin-resistant S. aureus both in vitro and in vivo. This work elucidates the mechanism of the oxidization strategy for enhancing the ROS production ability of PSs and offers a new guideline for the exploitation of AIE-active type I PSs.


Inner Mongolia University.
Instruments 1 H and 13 C NMR spectra were recorded with a Bruker ARX 500 NMR spectrometer using tetramethylsilane (TMS) as a reference. High resolution mass spectra (HRMS) were measured with a LCMS9030 spectrometer. UV-vis absorption spectra were recorded on a SHIMADZU UV-2600i spectrophotometer. Photoluminescence (PL) spectra were recorded on a HITACHI F-4700 fluorescence spectrophotometer. The absolute fluorescence quantum yield was measured using a Hamamatsu quantum yield spectrometer C11347-11 Quantaurus QY. Single crystal X-ray diffraction was performed on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation. The data were collected and processed using CrysAlisPro. Size distribution and zeta potential were analyzed on a dynamic light scattering (DLS) using an Omni NanoBrook. The cell viability was detected by CCK-8 kit, and the absorbance of each sample was measured at 450 nm using a microplate reader (BioTek). The bacterial fluorescence images were taken by inverted fluorescence S3 microscope (Nikon Ti2). The bacterial morphology was observed on a HITACHI S-4800 scanning electron microscope. The photographs of agar plate were taken by an automated colony counter (Shineso Icount33).

Density functional theory calculations
The density functional theory (DFT) method was used to optimize the molecular geometries of MPD and MPD-O in gaseous state at the level of B3LYP/6-31G(d). The frontier molecular orbitals of the dimers in crystal were calculated at the level of B3LYP/6-31G(d). The excited energies were calculated by the time-dependent density functional theory (TD-DFT) method at the level of wB97XD/def2TZVP. All the calculations were performed within Gaussian 09 software package. The SOCs between singlet and triplet states were given by PySOC 1 and atomic integrals in PySOC were calculated from MolSOC code developed by Sandro Giuseppe Chiodo et al. 2 IGM analysis 3,4 of weak interaction based on single crystal structure was conducted by using Multiwfn. 5 The corresponding structure and IGM isosurfaces were generated using VMD. 6
Then the hydrolysate was neutralized with 10 mL of 1 × PBS at pH 7.4, and kept in dark

ESR Analysis
ESR measurement was used to identify the type of ROS using 5-tert-butoxycarbonyl-5-methyl-

Bacteria Culture
A single colony of S. aureus, MRSA or E. coli on LB agar was transferred to 10 mL of LB liquid culture medium and grown for 10 h at 37 ℃ with a shaking speed of 220 rpm. Bacteria were harvested by centrifuging at 4000 rpm for 7 min and washed twice with PBS (pH = 7.4). After removal of the supernatant, the remaining bacteria were resuspended in PBS, and diluted to an optical density of 1.0 at 600 nm (OD600 = 1.0 with about 10 9 CFU mL -1 ).

Bacteria Staining and Imaging
After harvested by centrifugation, 1 mL of S. aureus or MRSA solution in PBS with a density of 2×10 8 CFU mL -1 were mixed with DAPD-O (20 μM), respectively. After dispersion with vortex, the samples were incubated at 37 °C with a shaking speed of 220 rpm for 2 h, respectively. To capture fluorescence images, 2 µL of stained bacteria solution was transferred to a piece of glass slide and then covered by a coverslip. The images were collected using an inverted fluorescence microscope. Capture conditions: PSs: λex = 352-402 nm and λem = 417-477 nm.

Antimicrobial Assay
Bacteria (S. aureus) at a density of ~10 7 CFU mL -1 were dispersed in the solutions containing MPD-O (0, 2, 5, 10 and 20 µM). Bacteria (S. aureus, E. coli or MRSA) at a density of ~10 7 CFU mL -1 were dispersed in the solutions containing DAPD-O (0, 2, 4, 6, 8 and 10 µM). These mixed solutions were then incubated at 37°C with a shaking speed of 220 rpm for 30 min. Next, the bacterial suspensions were exposed to white light irradiation for 40 min (100 mW cm -2 ) for phototoxicity test or were further incubated in the darkness at 37℃ for assessing the dark toxicity. Afterward, the samples were diluted to a density of ~10 2 CFU mL -1 with 1×PBS and spread on the LB agar plate, followed by culturing at 37 °C for 16 h before colony forming units (CFU) counting and taking photos.
Before the experiments, the cells were precultured until confluence was reached.

Live/dead staining assay
Followed by antimicrobial experiments, the bacteria were collected after irradiation and incubated with Calcein-AM (10 µM) and PI (10 µM) for 1 h. Then, the bacteria were washed one time with sterile PBS. The resulting bacterial suspension (2 μL) was added onto a glass slide, which was immobilized by a clean coverslip for characterization by inverted fluorescence microscope.

SEM analysis
Followed by antimicrobial experiments, the bacteria were collected after irradiation and fixed with 2.5% glutaraldehyde overnight. The glutaraldehyde was removed by centrifugation and the bacteria were washed with PBS for 2 times. Then the bacteria were dehydrated with a series S7 of graded ethanol/water solution (vethanol /vwater =10%, 30%, 50%, 70%, 80% 90%, 100%) for 15 min each. 2 μL of bacterial suspensions were added onto clean silicon slices followed by naturally drying in the air. The specimens were coated with Au before SEM analysis.

Histological Analysis
The wounds were histologically analyzed at day 8 post operation. Wound tissues were collected and fixed in 4% formaldehyde solution for 30 min. The pathological sections of wound tissues were analyzed by H&E staining. Histological images were taken by an inverted microscopy.

Biosafety Assessment
To further evaluate the safety of different treatments in vivo, blood samples were collected from mice with various treatments for complete blood panel analysis. White blood cell (WBC) counts, lymphocyte counts (LYMPH#), neutrophil counts (NEUT#), red blood cell (RBC), hemoglobin (HGB), and platelets (PLT) were measured.

Statistical Analysis
All data were expressed in this article as mean result ± standard deviation (s.d.). Statistically significant difference was evaluated by t-test, and statistical significance was considered as *p < 0.05, **p < 0.005 and ***p < 0.001 (n = 3).
The reaction was acidified by addition of 0.5 M aq. HCl (4 mL) and extracted with CH2Cl2.
The combined organics were washed with saturated aq. NaHCO3, dried over Na2SO4 and filtered. The resulting solution was concentrated under reduced pressure to provide a yellow residue. Recrystallization from toluene (0.5 mL/mmol) afforded the white solid of 2 (2.6 g, 65% yield). 1