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. 2023 Feb 11;16(2):275.
doi: 10.3390/ph16020275.

Curcumin-Functionalized Graphene Oxide Strongly Prevents Candida parapsilosis Adhesion and Biofilm Formation

Margherita Cacaci  1   2 , Damiano Squitieri  1 , Valentina Palmieri  3   4   5 , Riccardo Torelli  2 , Giordano Perini  4 , Michela Campolo  1 , Maura Di Vito  1 , Massimiliano Papi  4   5 , Brunella Posteraro  1   6 , Maurizio Sanguinetti  1   2 , Francesca Bugli  1   2
Affiliations

Curcumin-Functionalized Graphene Oxide Strongly Prevents Candida parapsilosis Adhesion and Biofilm Formation

Margherita Cacaci et al. Pharmaceuticals (Basel). .

Abstract

Candida parapsilosis is the major non-C. albicans species involved in the colonization of central venous catheters, causing bloodstream infections. Biofilm formation on medical devices is considered one of the main causes of healthcare-associated infections and represents a global public health problem. In this context, the development of new nanomaterials that exhibit anti-adhesive and anti-biofilm properties for the coating of medical devices is crucial. In this work, we aimed to characterize the antimicrobial activity of two different coated-surfaces, graphene oxide (GO) and curcumin-graphene oxide (GO/CU) for the first time, against C. parapsilosis. We report the capacity of GO to bind and stabilize CU molecules, realizing a homogenous coated surface. We tested the anti-planktonic activity of GO and GO/CU by growth curve analysis and quantification of Reactive Oxigen Species( ROS) production. Then, we tested the antibiofilm activity by adhesion assay, crystal violet assay, and live and dead assay; moreover, the inhibition of the formation of a mature biofilm was investigated by a viability test and the use of specific dyes for the visualization of the cells and the extra-polymeric substances. Our data report that GO/CU has anti-planktonic, anti-adhesive, and anti-biofilm properties, showing a 72% cell viability reduction and a decrease of 85% in the secretion of extra-cellular substances (EPS) after 72 h of incubation. In conclusion, we show that the GO/CU conjugate is a promising material for the development of medical devices that are refractory to microbial colonization, thus leading to a decrease in the impact of biofilm-related infections.

Keywords: biofilm; biofilm-related infections; graphene; natural anti-biofilm compounds.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Representative AFM image of GO; the scale bar is 1 µm. (B) Lateral size distribution of the flakes obtained through the analysis of three different images by ImageJ Fiji Software (Version 1.53 c).
Figure 2
Figure 2
(A) FTIR spectroscopy of GO and (B) GO/CU with characteristic peaks highlighted. The material was directly laid on the ATR crystal, and the spectra were recorded.
Figure 3
Figure 3
Growth curve of C. parapsilosis incubated with plastic (CTRL), GO, and GO/CU. O.D630 was monitored every hour for 18 h.
Figure 4
Figure 4
Intracellular ROS generation of C. parapsilosis cells in contact with plastic (control) surfaces, GO, and GO/CU after 6 h of incubation. Standard deviations of three independent experiments are represented by error bars. p values < 0.05 were considered significant: * < 0.05, ** < 0.005. RFU: relative fluorescent unit.
Figure 5
Figure 5
Adhesion assay. Candida cells were incubated with plastic, GO, and GO/CU for 30 min and two hours. Adherent cells were collected and enumerated by plating serial dilutions on Sabouraud agar plates and counting the CFU. Standard deviations of three independent experiments are represented by error bars. p values < 0.05 were considered significant: * < 0.05.
Figure 6
Figure 6
CV quantification of biofilm formation after 24 h on plastic, GO, and GO/CU. Biofilm biomass was stained with crystal violet, and then absorbance was read at 595 nm. Standard deviations of three independent experiments are represented by error bars. p values < 0.05 were considered significant: *** < 0.001.
Figure 7
Figure 7
Live and dead representative fluorescent images of Candida biofilm grown on a plastic surface (A) and GO (B) and GO/CU (C) surfaces for 24 h. Live cells are stained green (A1–C1), and dead cells are stained red (A2–C2). Images were merged (A3–C3) with ImageJ Fiji software. Images were taken using the Cytation 5 Cell Imaging Multi-Mode Reader (Agilent, Santa Clara, CA, USA). The scale bar is 100 µm.
Figure 8
Figure 8
Fluorescein diacetate assay (FDA). (A) Representative confocal microscopy images of Candida biofilm grown for 72 h on plastic (CTRL), GO, and GO/CU surfaces. Images were taken at 40X magnification. The scale bar is 100 μM. (B). The viability (percentage of control) of biofilm-associated cells was evaluated using FDA. Fluorescent intensities were determined by ImageJ Fiji software. Standard deviations of three independent experiments are represented by error bars. p values < 0.05 were considered significant: *** < 0.001.
Figure 9
Figure 9
Confocal microscopy representative images of Candida biofilm cells grown on plastic (A), GO (B), and GO/CU (C) for 72 h. Cells were stained with Syto 9 (A1–C1) and ConcA (A2–C2). Images were merged (A3–C3) with ImageJ Fiji software. The scale bar is 100 μM.
Figure 10
Figure 10
Fluorescence intensity analysis of Syto9/ConcA (percentage of the control) on the three different surfaces, plastic (control), GO, and GO/CU. Fluorescent intensity was determined by ImageJ Fiji software. Standard deviations of three independent experiments are represented by error bars. p values < 0.05 were considered significant: *** < 0.001.
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