Fig 1.
A schematic illustration of the synthetic route of the PLA-HA membrane and its antifungal applications in C. auris planktonic cells and biofilms, as well as the corresponding infected wound healing.
Fig 2.
Visual appearance and surface morphologies of PLA and PLA-HA nanofibrous membranes.
Scale = 5 μm, 2 μm and 0.5 μm from low magnification to high magnification in SEM images.
Fig 3.
In vitro antifungal activities and biosafety of PLA-HA and PLA.
(A) Spread plate results of C. auris growth under different conditions. The images from left to right represent PLA and PLA-HA were repeatedly used for four times. (B) SEM and TEM images of C. auris under different conditions. CM indicates the cytoplasmic membrane, CW represents the cell wall, N indicates the nucleus and L refers to lipid inclusion. (C) Corresponding survival rate results of C. auris with PLA and PLA-HA treatment (n = 3). (D) Hemolysis assay. The inset shows the image directly observed after adding PLA and PLA-HA for 2 h. Distilled water was used as a positive control, and PBS was used as a negative control. (E) Cell toxicity evaluation of PLA and PLA-HA on mouse fibroblast L929 cells for 24 h, 48 h and 72 h. Data are presented as the mean ± s.d.
Fig 4.
Biofilm formation and capacity of C. auris biofilm eradication.
(A) Observed C. auris biofilm formation at different times using Styo9 staining. Scale = 200 μm. (B) Growth curve of C. auris biofilm by the XTT reduction method. (C) Capacity of C. auris biofilm eradication of PLA and PLA-HA with and without illumination using the XTT reduction method. (D) Live/Dead staining of C. auris biofilms observed by CLSM. Scale = 10 μm. E. SEM images of C. auris biofilm with PLA and PLA-HA treatment. Data are presented as the mean ± s.d. n = 3.
Fig 5.
Antifungal efficacy and compatibility of PLA-HA in treating cutaneous C. auris infections.
(A)Schematic diagram of cutaneous wound infection with C. auris and treatment with PLA-HA. Rats were evaluated on the 3rd and 21st days. (B) Survival rate of C. auris in the infected wound sites treated with PLA-HA and PLA. (C) Corresponding wound healing rate of rats in different groups. (D) Representative photographs of wounds and fungal burden of PLA-HA and PLA treated rats on days 3 and day 21. (E) Infected skin wound tissue evaluated by H&E staining (day 21, scale = 100 μm), PAS staining (day 3, scale = 50 μm) and IHC of IL-6 (day 3, scale = 20 μm) after PLA-HA treatment. (F) H&E staining of lung, liver, spleen, kidney and heart in different groups after treatment (scale = 20 μm). Data are presented as the mean ± s.d.
Table 1.
The expression level of IL-6 in C. auris-infected tissue sites of different groups.
Fig 6.
ROS levels of PLA-HA mediated aPDT.
(A) The detected principle of intracellular ROS. (B) DCFH-DA detected by fluorescence microscopy (scale = 20 μm). (C) Fluorescence microscopy images of SOSG-stained C. auris (scale = 20 μm). (D) Changes in intracellular ROS levels after treatment with PLA-HA. (E) Absorbance of DPBF treated with PLA-HA. (F) UV-visible spectroscopic monitoring of the photooxidation of KI to I3- by PLA-HA. Data are presented as the mean ± s.d.
Fig 7.
Antifungal Mechanism of PLA-HA mediated aPDT.
(A) TUNEL, DAPI and Metacaspase detected by fluorescence microscopy. Scale = 10 μm. (B)Apoptosis of C. auris cells by staining with Annexin V-FITC and PI. (C) Mitochondrial membrane potential was evaluated using JC-1 staining. (D&E) Cytochrome C release from mitochondria to cytoplasm was assayed after treatment. Data are presented as the mean ± s.d.