Fig.1 Schematic illustration of Ru(II) supramolecular sonosensitizer-mediated sonodynamic therapy for fungal infection. (a) Design and synthesis of supramolecular sonosensitizers. (b) The mechanism and application of RuB-C12-mediated sonotherapy.#

Fig.2 Characterization of RuA-C0, RuA-C6, RuB-C6, and RuB-C12. (a) The chemical structures. (b) The partial 1H NMR (400 MHz, 298 K) spectra of the ligand (top), SCCs (middle), and acceptor (bottom). (c) Calculated and experimental ESI-TOF-MS spectra of RuA-C0 ([RuA-C0–4OTf]4+), RuA-C6 ([RuA-C6–4OTf]4+), RuB-C6 ([RuB-C6–4OTf]4+) and RuB-C12 ([RuB-C12–4OTf]4+).#

体外实验基于白念珠菌、光滑念珠菌和新型隐球菌等标准及临床菌株，系统评估了声动力学性能、生物被膜渗透及细胞毒性。实验显示，最优分子RuB-C12在超声触发下的活性氧生成量较传统声敏剂二氢卟吩（Ce6）提高3.0倍，对真菌PI的结合常数（$K_d$）达24.6 μM，在白念珠菌荧光检测灵敏度上比商业化荧光增白剂高出约45倍。超声激活下，RuB-C12表现出优异的抑菌浓度（白念珠菌、光滑念珠菌、新型隐球菌的MIC分别为2.5、1.25、0.625 μM），性能显著优于两性霉素B与氟康唑，并能清除90%以上的成熟生物被膜；针对正常哺乳动物细胞的选择性指数（SI）达10–20（远高于两性霉素B的SI < 5）。

Fig.3 Photophysical, sonodynamic, and sonocatalytic properties and PI recognition of supramolecular sonosensitizers. (a) UV–vis absorption and fluorescence emission spectrum of RuA-C0, RuA-C6, RuB-C6, and RuB-C12 in DMSO. (b) The stability of RuB-C12 under US irradiation examined by UV–vis absorption. (c) Fluorescent intensity changes of DCF at 525 nm generated by different compounds under ultrasound irradiation (1 W cm–2, 5 min). (d) Absorption spectra changes of ABDA at 399 nm generated by different compounds under US irradiation quantified by UV–vis absorption decrement (A/A0) at 399 nm of ABDA. (e) Fluorescent images of DCF under US irradiation (1 MHz, 50% duty cycle, 1 W cm–2, 5 min) with different compounds in different water fraction. (f) Plots of ln(A/A0) of NADPH at 339 nm for different time intervals. (g) UV–vis absorption spectra of RuB-C12 after adding different sterols and phospholipids. (h) ITC analysis of the interactions between RuB-C12 and PI. (i) Comparison of fungal cell detection sensitivity between RuB-C12 and Calcofluor White (CFW).#

Fig.4 In vitro evaluation of the antifungal activity of RuB-C12. (a) NIR-II fluorescence images of C. albicans or mammalian cells incubation with RuB-C12 by different treatments, Scale bar: 20 μm. (b) Zeta potential measurements of C. albicans treated with different concentrations of RuB-C12. (c) NIR-II fluorescence images of C. albicans biofilms. Scale bar: 200 μm. (d) Corresponding MIC values of RuB-C12 with/without US irradiation, AmB, and Flu against three yeasts. (e) Photographs of C. albicans colonies with different treatments (2.5 μM). Scale bar: 1 cm. (f) Killing kinetics of different compounds against actively growing C. albicans. (g) The survival rate of C. albicans biofilm after treatment with RuB-C12 and AmB, with or without US irradiation at 4 × MIC (n = 3, mean ± SD, ****P < 0.0001). (h) The selectivity index of different compounds against C. albicans calculated by the ratio of IC50 to MIC, respectively. (i) Cell viability of C. albicans incubation with RuB-C12 and US irradiation in the presence of PI with different concentrations. Darker color indicates high fungal growth.#

在体内与临床样本转化验证中，小鼠浅表伤口和鼻腔接种构建的深部肺部感染模型证实，RuB-C12结合近红外二区（NIR-II）荧光实现了深部肺组织成像（信噪比达12.0），超声治疗后肺部真菌负荷显著下降2.6个对数级，且无两性霉素B引起的肾毒性（肌酐升高）或主要脏器损伤。此外，在临床患者支气管肺泡灌洗液（BALF）样本中，该疗法同样实现了近乎完全的真菌清除，转录组测序进一步证实其通过协同破坏细胞膜完整性、诱发致死性脂质过氧化及线粒体功能障碍发挥疗效。

Fig.5 Mechanistic investigation of antifungal activity of RuB-C12. (a) Determination of the critical aggregation concentration (CAC) of PI by Nile Red (NR) (λem = 635 nm) in the presence of different concentrations of RuB-C12. (b) Fluorescence intensity of Disc3(5) probe in C. albicans changed in response to incubation with different concentrations of RuB-C12. (c) CLSM images of C. albicans with different treatments stained with Laurdan probe (50 μM). Scale bar, 20 μm. (d) TEM images of fungal cells coculture with RPMI 1640 medium or RuB-C12 (4 × MIC) for 2 h with US irradiation (1 W cm–2, 5 min). (e) 3D CLSM images of C. albicans biofilm with different treatments stained with DCFH probe (5 μM). Scale bar, 200 μm. (f) CLSM images of C. albicans in different treatments stained with JC-1 probe (2 μg/mL). Scale bar, 20 μm. (g) Intracellular NADPH and ATP levels in C. albicans with different treatments. (h) 3D CLSM images of C. albicans, C. glabrata and C. neoformans H99 biofilms with different treatments stained with AO/EB probes (10 μM). Scale bar, 200 μm. (i) Schematic diagram illustrating the mechanism of RuB-C12-mediated in C. albicans and biofilm.#

Fig.6 In vivo evaluation of the antifungal activity of RuB-C12. (a) Schematic diagram of the experimental protocol for pulmonary C. albicans infection treatment. (b) Real-time imaging of pulmonary C. albicans infection mice after intranasal administration of RuB-C12. (c) SBRs of NIR-II and visible-light fluorescence in the lungs of the mice (n = 3, mean ± SD, ***P < 0.001). (d) Representative images of lungs collected from mice with different treatments after 7 days. Scale bar: 2 cm. (e) Fungal loads in the lungs tissue with different treatment after 7 days (n = 3, mean ± SD, ****P < 0.0001). (f) Blood biochemistry analysis of mice with different treatments: CREA, μmol/L; BUN, mmol/L; ALT, U/L; AST, U/L; ALP, U/L; CK, U/L; LDH U/L; CKMB, U/L. Note: The normal reference range for CREA in mice is 18–71 μmol/L. (g) Volcano plot showing DEGs in PBS and RuB-C12 + US groups. (h) GO enrichment analysis of the gene functions of DEGs. (i) Scheme illustrating the application of RuB-C12 in clinical human bronchoalveolar lavage fluid (BALF). (j) Fungal burdens in BALF from Flu-, AmB-, and RuB-C12 + US-treated groups.#