A Membrane‐Targeted Photosensitizer Prevents Drug Resistance and Induces Immune Response in Treating Candidiasis

Abstract Candida albicans (C. albicans), a ubiquitous polymorphic fungus in humans, causes different types of candidiasis, including oral candidiasis (OC) and vulvovaginal candidiasis (VVC), which are physically and mentally concerning and financially costly. Thus, developing alternative antifungals that prevent drug resistance and induce immunity to eliminate Candida biofilms is crucial. Herein, a novel membrane‐targeted aggregation‐induced emission (AIE) photosensitizer (PS), TBTCP‐QY, is developed for highly efficient photodynamic therapy (PDT) of candidiasis. TBTCP‐QY has a high molar absorption coefficient and an excellent ability to generate 1O2 and •OH, entering the interior of biofilms due to its high permeability. Furthermore, TBTCP‐QY can efficiently inhibit biofilm formation by suppressing the expression of genes related to the adhesion (ALS3, EAP1, and HWP1), invasion (SAP1 and SAP2), and drug resistance (MDR1) of C. albicans, which is also advantageous for eliminating potential fungal resistance to treat clinical infectious diseases. TBTCP‐QY‐mediated PDT efficiently targets OC and VVC in vivo in a mouse model, induces immune response, relieves inflammation, and accelerates the healing of mucosal defects to combat infections caused by clinically isolated fluconazole‐resistant strains. Moreover, TBTCP‐QY demonstrates excellent biocompatibility, suggesting its potential applications in the clinical treatment of OC and VVC.


Figure S5 .
Figure S5.Images of the plasma membrane in HEK-293 cells stained with TBTCP-QY.The red channel used a 561 nm laser and a 570-620 nm emission filter (scale bar: 20 μm).

Figure S6 .
Figure S6.CLSM images of C. albicans stained with TBTCP-QY.The red channel used a 561 nm laser and a 570-620 nm emission filter (scale bar: 60 μm).

Figure S7 .
Figure S7.CLSM images of C. albicans counterstained with DAPI and TBTCP-QY.The red channel used a 561 nm laser and a 570-620 nm emission filter, and the blue channel used a 405 nm laser and a 425-475 nm emission filter (scale bar: 20 μm).

Figure S8 .
Figure S8.CLSM images of C. albicans stained with TBTCP-QY for different time.The red channel used a 561 nm laser and a 570-620 nm emission filter (scale bar: 10 μm).

Figure S9 .
Figure S9.FESEM images of C. albicans incubated with different concentrations of TBTCP-QY with or without light irradiation for 15 min (80 mW cm -2 ).The red arrows indicate deformed or broken fungal structures (scale bar: 10 μm).

Figure S10 .
Figure S10.The absorbance value of C. albicans biofilm at 595 nm with different treatments and then stained with crystal violet.Data are expressed as the mean ± SD of 3 replicates.Statistical significance between every two groups was calculated via one-way ANOVA.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Figure S11 .
Figure S11.Fluorescence images of the plasma membrane of C. albicans in biofilms stained with TBTCP-QY.The red channel used a 561 nm laser and a 570-620 nm emission filter (scale bar: 20 μm).

Figure S12 .
Figure S12.Evaluation of reactive oxygen species production in biofilms.A) CLSM 3D images of biofilms (scale bar: 60 μm) and B) quantification of the corresponding fluorescence intensity according to COMSTAT 2.0 for five random sights of C. albicans biofilms.Biofilms were stained with DCFH, incubated without/with 5 µM of TBTCP-QY and then irradiated with light (80 mW cm -2 ).The green channel uses a 488 nm laser and a 515-550 nm emission filter.Data are shown as mean ± SD.

Figure S13 .Figure S14 .
Figure S13.3D images of biofilms treated with TBTCP-QY or CHX and white light irradiation and then stained with a Live & Dead TM activity/cytotoxicity analysis kit (UElandy).The green channel used a 488 nm laser and a 515-550 nm emission filter.The red channel used a 561 nm laser and 570-620 nm emission filter (scale bar: 20 μm).

Figure S15 .
Figure S15.Representative images of OC mice from different treatment groups (TBTCP-QY+L, CHX and PBS) after 7 days (n = 5 mice in each group).Mice without C. albicans infection were used as the Control group.

Figure S16 .
Figure S16.Plate images of bacteria (gray, LB agar) and fungi (green, CCM agar) from vaginal washes taken in vivo VVC model mice on Day 1 after treatment.

Figure S17 .
Figure S17.Plate images of the samples from VVC mice infected with strain A) HX0819 and strain B) SC5314 on Day 5 with different treatments.Data are shown as mean ± SD.

Figure S19 .
Figure S19.Photodynamic antifungal effect of TBTCP-QY on clinical sample.A) Schematic diagram of the experimental procedure for taking clinical samples and treating them using TBTCP-QY and white light irradiation.B) LB agar images of clinical samples (BV) before and after treated with TBTCP-QY and light irradiation (0.8 μM, 80 mW cm -2 ).C) LB agar images of clinical samples (BV+VVC) before and after treated with TBTCP-QY and light irradiation (0.8 μM, 80 mW cm -2 ).

Figure S20 .
Figure S20.Survival rate A) S. aureus, B) MRSA and C) E. coli after treatment of TBTCP-QY,

Figure S21 .
Figure S21.PDI of C. albicans HX0819 after ten consecutive cycles treatment with 0.3 μM TBTCP-QY for 15 min and irradiation with white light for 15 min (80 mW cm -2 ) followed by staining with a Live & Dead TM activity/cytotoxicity analysis kit (UElandy).The green channel used a 488 nm laser and a 515-550 nm emission filter, and the red channel used a 561 nm laser and 570-620 nm emission filter (scale bar: 20 μm).

Figure S22 .Figure S23 .
Figure S22.Photodynamic antimicrobial effect of TBTCP-QY on drug resistant C. albicans HX0819.A) Photodynamic inactivation of C. albicans HX0819 after ten consecutive cycles of treatment with 0.8 μM TBTCP-QY for 15 min and irradiation with white light for 15 min (80 mW cm -2 ) followed by staining with a Live & Dead TM activity/cytotoxicity analysis kit (Everbright, USA).The green channel used a 488 nm laser and a 515-550 nm emission filter, and the red channel used a 561 nm laser and 570-620 nm emission filter (scale bar: 20 μm).B) mRNA expression of MDR1 before and after C. albicans HX0819 was treated with ten PDI cycles.Untreated fungi were used as control.Data are shown as mean ± SD.

Table S2 .
Absolute expression of immune factors in mice.

Table S3 .
Patients clinical sample information.

Table S4 .
Summary of patients' information.

Table S5 .
Zeta potential results of C. albicans and HEK-293 cells in PBS solution pretreated with or without 5 μM TBTCP-QY.