BODIPY-Based NIR Trackers with Acidity-Driven Amphiphilicity for STED Super-Resolution Imaging of Lysosomal Membranes.

Although super-resolution imaging provides a great opportunity to disclose the structures of living cells at the nanoscale level, resolving the structural details of organelles is highly dependent on the targeting accuracy and photophysical properties of fluorescence trackers. Herein, we report a series of ultrabright and photostable trackers of lysosomal membranes for super-resolution imaging using stimulated emission depletion microscopy (STED). These trackers are composed of lipophilic NIR BODIPY derivatives and ionizable tertiary amines. This structural feature enables accurate targeting of the lysosomal membrane through the formation of transient amphiphilicity driven by the acidity in the lysosome. As a representative, Lyso-700 is applied for STED-based super-resolution imaging of the lysosomal membrane of living macrophages. By use of Lyso-700, the interaction details between lysosomes of macrophages and fungi are visualized. Overall, these trackers display great potential as advanced lysosome trackers and merit further evaluation for lysosome-related studies.

Adoptive macrophage directed photodynamic therapy of multidrug-resistant bacterial infection.

Multidrug-resistant (MDR) bacteria cause severe clinical infections and a high mortality rate of over 40% in patients with immunodeficiencies. Therefore, more effective, broad-spectrum, and accurate treatment for severe cases of infection is urgently needed. Here, we present an adoptive transfer of macrophages loaded with a near-infrared photosensitizer (Lyso700D) in lysosomes to boost innate immunity and capture and eliminate bacteria through a photodynamic effect. In this design, the macrophages can track and capture bacteria into the lysosomes through innate immunity, thereby delivering the photosensitizer to the bacteria within a single lysosome, maximizing the photodynamic effect and minimizing the side effects. Our results demonstrate that this therapeutic strategy eliminated MDR Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB) efficiently and cured infected mice in both two models with 100% survival compared to 10% in the control groups. Promisingly, in a rat model of central nervous system bacterial infection, we performed the therapy using bone marrow-divided macrophages and implanted glass fiber to conduct light irradiation through the lumbar cistern. 100% of infected rats survived while none of the control group survived. Our work proposes an efaficient and safe strategy to cure MDR bacterial infections, which may benefit the future clinical treatment of infection.

Polarity-Sensitive and Membrane-Specific Probe Quantitatively Monitoring Ferroptosis through Fluorescence Lifetime Imaging.

As a new form of regulated cell death, ferroptosis is closely related to various diseases. To interpret this biological behavior and monitor related pathological processes, it is necessary to develop appropriate detection strategies and tools. Considering that ferroptosis is featured with remarkable lipid peroxidation of various cell membranes, it is logical to detect membranes' structural and environmental changes for the direct assessment of ferroptosis. For this sake, we designed novel polarity-sensitive fluorescent probes Mem-C1C18 and Mem-C18C18, which have superior plasma membrane anchorage, high brightness, and sensitive responses to environmental polarity by changing their fluorescence lifetimes. Mem-C1C18 with much less tendency to aggregate than Mem-C18C18 outperformed the latter in high resolution fluorescence labeling of artificial vesicle membranes and plasma membranes of live cells. Thus, Mem-C1C18 was selected to monitor plasma membranes damaged along ferroptosis process for the first time, in combination with the technique of fluorescence lifetime imaging (FLIM). After treating HeLa cells with Erastin, a typical ferroptosis inducer, the mean fluorescence lifetime of Mem-C1C18 displayed a considerable increase from 3.00 to 4.93 ns, with a 64% increase (corresponding to the polarity parameter Δf increased from 0.213 to 0.232). Therefore, our idea to utilize a probe to quantitate the changes in polarity of plasma membranes proves to be an effective method in the evaluation of the ferroptosis process.