Fig 1.
Characterization of in-house synthesized GNPs and PS conjugation onto their surface.
(A) Characterization of in-house synthesized gold nanoparticles. (a) Ultraviolet-visible- near infrared spectra of gold nanoparticles synthesized by exposing various amounts of Aloe vera extract to a fixed volume of HAuCl4 solution (10−3 M), keeping the final volume of reaction mixture 10 ml that was made up for each sample by increment in Aloe vera extract content for 24 h. Absorbance is seen to be increasing exhibiting two major peaks, a larger one at 540 nm, the characteristic peak of GNPs. (b) Representative TEM image of gold nanoparticles synthesized using Aloe vera leaf extract exhibiting a large population of spherical gold nanoparticles of approx. 10 nm size. (c) Nanophox particle analysis of synthesized gold nanoparticles. (B) Ultraviolet-visible absorption spectra of gold nanoparticle-photosensitizer conjugate normalized to the maximum absorbance intensity. GNP has signature spectra at 540 nm due to surface plasmon resonance. MB shows absorption peaks at 662 nm and 613 nm. Conjugation of MB to GNP led to appearance of a small peak at 540 nm, intrinsic feature of GNP, which was absent in pure MB spectrum (a). TB spectrum enumerates peaks at 625 nm and 585 nm. GNP-TB conjugate is observed to exhibit an additional shoulder at 540 nm indicating conjugation of GNP to TB (b). Spectrum obtained for the mixture of GNP-MB and GNP-TB (c).
Fig 2.
Transmission electron microscopic images of GNP, GNP-TB and GNP—MB.
(A) Biomimetically synthesized naked spherical colloidal GNPs (B) MB-conjugated GNPs (C) TB-conjugated GNPs. Coating of PSs around GNPs is indicated by black arrows.
Fig 3.
Microscopic images revealing photodynamic inhibition of C. albicans biofilm grown on plastic coverslips.
(A) Untreated control image of the C. albicans biofilm (B) Biofilm treated with naked GNPs (C) Biofilm exposed to MB (D) Biofilm after treatment with TB (E) GNP-MB (F) GNP-TB treated biofilms (G) GNP-MB and GNP-TB combination exposed C. albicans biofilm.
Fig 4.
SEM micrographs showing marked inhibition of C. albicans biofilm by GNP-PS (GNP-MB and GNP-TB) combination mediated PDT.
(A) Untreated control image of the C. albicans biofilm (B) Naked GNPs treated biofilm (C) C. albicans biofilm treated with unconjugated MB (D) Biofilm exposed to TB (E) Biofilm treated with GNP-MB and (F) GNP-TB treated biofilm (G) GNP-MB and GNP-TB combination mediated reduction in biofilm.
Fig 5.
Photodynamic effect of GNP-MB and GNP-TB combination on C. albicans biofilm and planktonic cells.
Effect of GNP-MB and GNP-TB alone and in combination against biofilm development (A) and mature biofilm (B). Growth percentage was analyzed by comparing relative metabolic activity (RMA) obtained through XTT assay taking untreated control as 100%. Photodynamic inactivation of planktonic C. albicans in vitro (C). C. albicans yeast cells incubated with GNP-PS complexes, after being irradiated with respective light source were picked (50 μl) and plated onto YPD agar plates for counting CFU. The data represent the mean of three determinants ± SD (Standard Deviation) and are representative of three different experiments (i.e, the experiment was done in triplicate) with similar observations. Statistically groups were compared with control using ANOVA (Analysis of Variance) with the Holm—Sidak test. P values <0.05 were considered statistically significant. GNP-MB+GNP-TB vs Control, P<0.005.
Fig 6.
Uptake of GNP-PS conjugates by activated macrophages.
(A) Macrophages exposed to MB (B) TB treated macrophages (C) GNP-MB conjugate entrapped by macrophages (D) GNP-TB complex engulfed by macrophages (E) Macrophages are seen with GNP-MB and GNP-TB entrapped within. Upper panel shows fluorescent images and lower panel exhibits the corresponding brightfield images.
Fig 7.
GNP-PS conjugate mediated killing of C. albicans inside macrophages and reduction of fungal burden in skin C. albicans infection.
(A) C. albicans yeast cells and hyphal filaments are seen to be harbouring the intracellular niche of macrophage. (B) GNP-MB and GNP-TB mixture is exhibited to be the best amongst various formulations taken in inhibiting C. albicans residing within the macrophages. (C) PDT employing GNP-MB and GNP-TB conjugates result in reduced fungal burden in skin C. albicans infection. After a total light exposure of 21.6 J/cm2, GNP-PS complex treated wounds were excised aseptically. The homogenate was plated onto YPD agar plates to count the CFU. The combination of GNP-MB and GNP-TB, in a manner similar to in vitro results, depleted the fungal burden in the skin most efficiently when compared to other formulations. Data are representative of three independent experiments ± SD values. P values<0.05 were considered significant. GNP-MB+GNP-TB vs Control, P<0.005.
Fig 8.
Representative images of mouse skin infected with C. albicans and stained with periodic acid—Schiff; X400.
(A) Untreated control, showing exuberant growth (B) Naked GNPs, showing only mild response to treatment (C) MB treated group and (D) TB exposed group, exhibiting better response to treatment than GNPs (E) GNP-MB group and (F) GNP-TB group, showing inhibition on the growth and hyphal population is also observed to be highly decreased in comparison to control. (G) Combination of both GNP-MB and GNP-TB showing significant inhibition on the growth. The arrows indicate the population of C. albicans yeast cells and hyphae penetrating the skin tissue.
Fig 9.
Sagittal cut of the tongue dorsum of mouse oral candidiasis model.
Representative images of mouse tongue dorsum infected with C. albicans and stained with periodic acid—Schiff; X400. (A) Healthy control, showing numerous elongated papillae. (B) Untreated control showing numerous yeast cells and hyphal filaments inhabiting the tongue papillae (C) MB treated group and (D) TB treated group showing mild response to treatment (E) Mice tongue treated with GNP-MB and (F) GNP-TB treated group, showing comparatively better inhibition of hyphal population than control or unconjugated PS treated groups (G) Combination of both GNP-MB and GNP-TB showing marked inhibition on the growth of yeast cells as well as hyphae. The population of C. albicans yeast cells and hyphae penetrating the tongue dorsum is indicated by the arrows.