Therapeutic Potential of Green Synthesized Gold Nanoparticles Using Extract of Leptadenia hastata against Invasive Pulmonary Aspergillosis

Abstract

Gold nanoparticles are widely used in the biomedical field for the treatment of several diseases, including cancer, inflammatory diseases, and immune system disorders, due to their distinctive physicochemical characteristics. In this study, we investigated the therapeutic potential of green synthesized gold nanoparticles using ethanolic leaf extract of Leptadenia hastata (LH-AuNPs) against invasive pulmonary aspergillosis (IPA) in mice. UV/visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and zeta potential were used to characterize the biofabricated LH-AuNPs. Antifungal activity of LH-AuNPs was determined by MTT assay, (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), time-kill assay, and radial growth inhibition. TEM and SEM were used to examine the mode of the antifungal action of LH-AuNPs. The in vivo activity of LH-AuNPs against IPA was studied using a well-established IPA mouse model. LH-AuNPs excreted antifungal activity against Aspergillus fumigatus with MIC 64 µg/mL and inhibited the radial growth of A. fumigatus by 30% compared to the control. LH-AuNPs caused distortion and collapse of fungal hyphae and deterioration of cell walls. Interestingly, LH-AuNPs did not display any cytotoxicity on cultured primary bone marrow stem cells (BMSCs) or A549 human lung cell line in vitro at MIC concentration. IPA mice treated with LH-AuNPs displayed significant lung tissue repair without any in vivo cytotoxicity. LH-AuNPs administration showed significant suppression of fungal burden and gliotoxin production in the lung. In addition, LH-AuNPs inhibited IPA-induced pro-inflammatory cytokines production, including interleukin-1 (IL-1), interleukin-17 (IL-17), and tumor necrosis factor-alpha (TNF-α), and reduced oxidative stress in lung. In conclusion, our data provide LH-AuNPs as a novel nanoparticle therapy for IPA.

Keywords: A. fumigatus; IPA; Leptadenia hastata; aspergillosis; gold nanoparticles.

Figure 1

Biosynthesis of LH-AuNPs using L. hastate leaf extract. (A) The biosynthesis of LH-AuNPs was performed by combining L. hastate leaves powder (10 g) with 50 mL of 95% ethanol for 24 h at 37 °C in a 200 mL Erlenmeyer flask; 5 mL of the plant extract was added to 1 mM aqueous HAuCl4 solution (45 mL). The solution changed color from pale yellow to vivid ruby-red, signifying the formation of AuNPs. (B) TEM image of LH-AuNPs showed spherical or hexagonal shapes with lattice fringes s with sizes ranging from 5 to 30 nm.

Figure 2

Confirmation of biosynthesized LH-AuNPs. (A) UV–Vis spectrum of LH-AuNPs. Different ratios give different absorption peaks, but the most extreme peak with maximum absorbance was recorded 1:2 on 544 nm. (B) XRD spectrum recorded for LH-AuNPs showed clear peaks of cubic phases at 38.2 (111), 44.3 (200), 64.9 (220), 77.5 (311), and 81.5 (222), which confirms the crystalline nature of AuNPs. (C) FTIR spectrum of LH-AuNPs exhibited two peaks related to OH/NH and C=O groups. The presence of OH group could be ascribed to peak at 3444.6 cm⁻¹. The peak of 1732.0 corresponded to C=O group. (D) EDX spectrum of LH-AuNPs shows strong signals in the gold region and confirms the formation of gold nanoparticles. A strong peak was displayed around 2.40 keV, which is the characteristic of gold nanoparticles.

Figure 3

Antimicrobial potential of LH-AuNPs. (A) Inverted microscope images of A. fumigatus treated with DMSO, LH-AuNPs, and AMB at their MIC values. Visual alterations in mycelial growth are obvious at three different treatments. (B) Radial growth of A. fumigatus was inhibited, where LH-AuNPs reduced the relative radial growth of A. fumigatus by 30% compared to the control. (C) Pigment formation defects after LH-AuNPs treatment. The LH-AuNPs treated colonies lacked green pigmentation, signifying they formed few conidia. (D) Time–kill curves of A. fumigatus following exposure to LH-AuNPs and AMB. Values are mean ± SD of three independent experiments.

Figure 4

Electron microscopy photographs of A. fumigatus after treatment with LH-AuNPs. (A) SEM images of A. fumigatus treated with saline (a,b) and LH-AuNPs (c,d) (64 μg/mL). Black arrows specify pitting and tearing destruction to the cell wall. White arrows show penetration of cell wall into the cytoplasm. Bar = 5 μm. (B) TEM images of A. fumigatus treated with saline (a,b) showing normal growth of A. fumigatus hyphae and treated with LH-AuNPs (c,d) showing reticular accumulations on the cell wall on the outer fibrillar layer (arrows) (c) and the outer fibrillar layer has a lattice-like structure that is thready (thick arrows). The inner fibrillar layer is not consistently observable (thin arrow) (d).

Figure 5

LH-AuNPs repair lung tissue damage in IPA mice. (A) Cytotoxicity of LH-AuNPs on human lung cancer cell line, A549, and primary mBMSCs. The dose-dependent effect of LH-AuNPs on cell viability was measured by MTT assay after 48h of treatment. Values are mean ± SD of three independent experiments (** p < 0.005, compared to control non-treated cells). (B) Histological analysis of lung tissues (3 days post-LH-AuNPs treatment) from control, IPA-non-treated, and IPA-treated mice with LH-AuNPs. Sections stained with H & E (a) and periodic acid–Schiff (PAS) (b). Extensive fungal growth and tissue damage are evident in the non-treated IPA mice. Arrows indicate fungal balls with great density fungi and proliferating hyphae, while there is a lack of fungal balls and hyphae in the lungs of animals with LH-AuNPs treatment.

Figure 6

LH-AuNPs suppress fungal burden and gliotoxin production in lung of IPA mice. In vivo cytotoxicity of LH-AuNPs in IPA mice. (A) H & E histological sections of liver and kidney from control, IPA-non-treated, and IPA-treated mice with LH-AuNPs. (B) Serum biochemical markers of liver function (a) AST and ALT and (b) renal function, including urea and creatinine. Biochemical analysis was performed after 3 days of treatment with LH-AuNPs. (C) Effect of LH-AuNPs on fungal load in lung homogenate of IPA mice. (D) Measurements of lung gliotoxin concentration in IPA mice after 3 days of LH-AuNPs. Values are expressed as means ± SD (n = 10 mice/group) (** p < 0.005, compared to control non-treated mice).

Figure 7

Inhibitory effect of LH-AuNPs on pro-inflammatory cytokines production and oxidative stress in IPA mice. Measurements of inflammatory cytokines, (A) TNF-α, (B) IL-1, and (C) IL-17 after 3 days of LH-AuNPs treatment in IPA mice. Effect LH-AuNPs on the antioxidant enzymes production, including (D) CAT, (E) SOD, and (F) MDA in the lung of IPA mice after 3 days of LH-AuNPs treatment. Data are expressed as means ± SD (n = 10 mice/group). (* p < 0.05 and ** p < 0.005, compared to control non-treated mice).