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

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.


Introduction
The incidence of life-threatening mycotic infections caused by Aspergillus species and other fungi has increased in the past few years [1,2]. A. fumigatus is one of the most common fungal pathogens that can cause a variety of diseases, such as invasive pulmonary aspergillosis (IPA), aspergilloma, and allergic syndromes [3]. The clinical spectrum of patients who are at high risk for IPA is extensive. Patients getting allogeneic bone marrow transplants and patients with hematological malignancy have a higher risk of IPA [4].
Owing to the complications in treating IPA, it remains challenging to increase novel insights into the distribution of infection. The antifungal drugs used for the treatment of IPA are limited due to the eukaryotic nature of fungal cells; they have only a limited set of specific targets that do not overlay with their mammalian counterpart. The currently available antifungal agents have several associated problems. Amphotericin B (AMB) can cause severe adverse effects because of its nephrotoxicity [5]. Although lipid preparations of AMB have decreased nephrotoxicity, renal injury is still detected, and infusion-related toxicity might be debilitating. Itraconazole is not absorbed in adequately higher amounts to be therapeutic and might interact harmfully with an extensive spectrum of drugs [6].
Therefore, there is a critical necessity for the development of novel antifungal drugs with novel modes of action [7]. Gold nanoparticles have been the focus of increasing interest in many biomedical applications due to their mechanical, chemical, thermal, optical, and biocompatibility properties. These applications include the use of AuNPs in diagnostic tracers, biosensing, photothermal and radiotherapy, and cell imaging [8][9][10]. In addition, the approval of using AuNPs by the Food and Drug Administration (FDA) increased the applications of AuNPs as therapeutic agents in nanomedicine, such as cancer therapy and drug carriers [11,12].
Green synthesis of nanoparticles using biological extracts has been developed to provide large-scale production, where mild reaction conditions without additional reducing agents and stabilizers were applied that minimize the hazardous wastes and reduce the costs of nanoparticle production [13]. We have recently reported that the green synthesis of silver nanoparticles (AgNPs) using plant extracts could effectively produce nanoparticles with antimicrobial effects against C. albicans and A. fumigatus in vitro and in vivo [14,15].
L. hastata is a member of the family Asclepiadaceae, used as a vegetable, and is considered a scarce food due to its higher content of beneficial nutrients [16,17]. It is used in herbal medicine to treat diabetes [18] and has anti-inflammatory and antitrypanosomal activities [19]. It is also applied as an antimicrobial agent against several fungal and bacterial species [20,21]. In this study, we explore the antifungal action of biosynthesized AuNPs using leaf extract of L. hastata against A. fumigatus in vitro and in vivo. LH-AuNPs showed antifungal activity against A. fumigatus in vitro and therapeutic effect for IPA in vivo via reducing fungal burden, gliotoxin production, and IPA-induced pro-inflammatory cytokines.

Preparation of Plant Extract
L. hastata was identified taxonomically and authenticated by the Department of Botany and Microbiology, Cairo University, where a voucher specimen was deposited (Voucher number N8F5). Then, 10 g of dried L. hastata leaves powder was suspended in 50 mL of 95% ethanol for 24 h at 37 • C. The extract was filtered and evaporated by a rotary vacuum evaporator at 40 • C and stored at 4 • C.

Green Synthesis of LH-AuNPs
Five mL of the plant extract was added to 45 mL of 1 mM aqueous HAuCl4 solution. The change of color from pale yellow to vivid ruby-red demonstrates the reduction of AuCl 4 and the formation of AuNPs. The suspension was centrifuged at 3500 rpm for 10 min to remove the unreacted plant extract. The biosynthesized nanoparticles were collected by centrifugation at 12,000 rpm for 20 min and purified by washing with sterile distilled water to obtain nanoparticles in pellet form. The purified AuNPs were then suspended in distilled water for further study.

Characterization of LH-AuNPs
The reduction of gold ions was confirmed by measuring the UV-vis spectrum of the reaction mixture. The spectral analysis was done using a UV-1602 Double Beam UV/Vis spectrophotometer at a resolution of 1 nm from 300 nm to 800 nm. The selected ratio was allowed to freeze-dry for further characterizations. The crystalline pattern of powdered AuNPs was recorded by XDL 3000 powder X-ray diffractometer. The Fourier transform infrared (FTIR) spectra were performed on a Perkin Elmer spectrum instrument at a resolution of 4 cm −1 in KBr pellet. The morphology, purity, structure, and elemental distribution of the LH-AuNPs were observed by transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). TEM images were obtained by placing droplets of the purified NPs suspended in water on a carbon-coated copper grid and drying them at room temperature before microscopic analysis. EDX was done by Energy Dispersive X-ray analysis "EDX". A double-sided carbon coated glass cover slip was used, the lower side of which was used to be fixed to the stab, while, on upper side, sample was loaded and was examined by EDX (Oxford, UK, Model No. INCA 200). The zetapotential measurements were performed with a Zetasizer Nano ZS (Malvern Instruments) in a disposable cell at 25 • C, using Zetasizer 7.01 software. Zeta potential is used to study AuNPs stability. These measurements were carried out first 2-3 h after the LH-AuNPs were synthesized and then once a week for 3 weeks. Between measurements, all LH-AuNPs suspensions were kept frozen at −18 • C. The suspension was allowed to melt and equilibrate at room temperature for 2 h prior to measurement. Straight away after the measurements were taken, the extracts were frozen again.

Microorganism and Culture Conditions
A strain of A. fumigatus was formerly isolated by our group from an immunocompromised patient with IPA [14]. Fungal suspension was prepared by cultivating A. fumigatus on Peptone Yeast Extract Agar (PYG) medium for 5 days at 35 • C, and the conidia were harvested as described previously [22].

Antifungal Susceptibility Test
Antifungal assay was carried out by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) method with slight modifications [23]. The conidia were counted by using a hemocytometer, adjusted to a density of 10 6 CFU/mL; 96-well flatbottom plates were used, and 90 µL of RPMI 1640 and 100 µL from LH-AuNPs were added into the first wells. Then, LH-AuNPs were serially diluted to final concentrations ranging from 2 to 256 µg/mL. In each well of the column, aliquots of 10 µL of the A. fumigatus inoculum were dispensed. A positive control was prepared in the same way with the standard antifungal drug amphotericin B (AMB). After 24 h of incubation, 20 µL of an MTT was added to each well. The plates were then incubated at 37 • C for 48 h. After incubation, the formazan assay product was extracted, and the optical density at 540 nm was measured. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the drug that resulted in 100% visual inhibition of growth. For the determination of radial growth, the spores of A. fumigatus were added to the center of potato dextrose agar (PDA) plates containing sub-MIC of LH-AuNPs (32 µg/mL). DMSO was used as negative control. The plates were incubated at 35 • C, and growth of A. fumigatus was observed after 7 days. The micrographs were obtained using an Olympus Inverted Microscope IX50 equipped with a Lumenera Infinity camera (Olympus Corporation, Tokyo, Japan).

Pigment Inhibition
Conidial suspensions of A. fumigatus (1 × 10 5 ) were inoculated in PDA plates containing sub-MIC of LH-AuNPs (32 µg/mL) and incubated at 35 • C. After 72 h, the morphological examination of the colonies was performed.

Time-Kill Curve Studies
Briefly, 10 mL of A. fumigatus conidial suspension (1 × 10 5 conidia/mL) was added to 10 mL of RPMI-1640 medium (negative control) with AMB (positive control) or LH-AuNPs. Cultures were agitated at 37 • C. At different time intervals (0, 4, 8, 12, 16, and 24 h), a sample of 0.1 mL was removed from each test suspension, spread on PDA plates, and then incubated at 37 • C for 48 h. The time-kill curves were made by plotting the colonyforming units (CFU) per milliliter surviving at each time interval in the presence of various antimicrobial drugs. The LH-AuNPs-treated A. fumigatus mycelia sections were collected, fixed with formaldehyde, washed with phosphate buffer solution, dehydrated with an alcohol solution, and then submitted to critical point drying as described [24]. The fungal mycelia were prepared for SEM using JEOL (JSM6380 LA) instrument.

Transmission Electron Microscopy (TEM)
A. fumigatus mycelia treated with LH-AuNPs were observed using TEM. Saline-treated mycelia were used as a control. All samples were infiltrated with 1:1 then 1:2 ratios of ethanol to resin in a vacuum overnight. The samples were then fixed and allowed to evacuate overnight, then placed in an oven to polymerize for four days. The samples were trimmed and thin sectioned. The sections were post-stained with 7.5% uranyl acetate and Reynolds's lead citrate. TEM images of the samples were taken using the JEM-1210 TEM instrument (JEOL USA Inc., Peabody, MA, USA) and operated at 90 kV [25].

In Vivo Experiment Design
The procedure of in vivo IPA experiment was approved by the Cairo University, Faculty of Science Institutional Animal Care and Use Committee (IACUC) (Egypt), (CUFS/F/10/13). Swiss albino mice (10 weeks) were obtained from the animal house, National Research Center. Mice were housed at a controlled temperature of 25 ± 2 • C and 12 h dark/light cycle with a standard diet and water ad libitum. Thirty male mice were grouped into 3 groups (n = 10), and neutropenia was induced by a single intraperitoneal (ip) administration of cyclophosphamide (150 mg/kg) as described previously [26]. Three days post neutropenic induction, 20 of the neutropenic mice were infected with conidia of A. fumigatus (1 × 10 5 ) (administered by intranasal instillation), as described [27]. Mice groups were assigned as follows: group 1 (control): control mice injected intravenously with Hank's Balanced Salt Solution (HBSS); group 2 (IPA control): IPA mice intravenously injected with HBSS after 24 h of fungal inoculation; group 3 (LH-AuNPs group): IPA mice intravenously injected with one injection of LH-AuNPs (0.5 µg/g) after 24 h of fungal inoculation [28].

Measurement of Fungal Burden in Lung Tissue
Lungs were dissected from IPA mice after euthanization, weighed, and homogenized in 2 mL of sterile saline with sterile tissue grinders. Numbers of CFU were determined by performing serial dilutions of the homogenized tissue in sterile saline and plating 50 g of lung homogenate on 110 mm diameter potato dextrose agar plates. Plates were incubated at 37 • C, and numbers of CFU were counted at 24 and 48 h.

Measurement of Gliotoxin
Lung tissues were soddened in plastic bags and mixed with 5 mL of water. The samples were then homogenized, and 10 mL HCl was added to homogenates on a shaker for 30 min. Supernatants of cell cultures were exposed to solid-phase extraction by loading 1 mL of each sample solution onto an Oasis HLB cartridge (Waters Corporation, Milford, MA, USA), which was conditioned with 2 mL of methanol-water mixture. The cartridge was then washed with 1 mL of 5% methanol in water, and the analyte was eluted with 1 mL of absolute methanol. For HPLC analysis, the extract was evaporated to dryness at 50 • C; the residue was added to 0.2 mL methanol and used for the gliotoxin measurement according to the method of [29]. Briefly, 20 µL of the sample was injected at a flow rate of 1 mL min −1 . Gliotoxin was eluted from the HPLC column after 20 min, and the concentrations were measured by interpolation from a calibration curve (25-1000 ng mL −1 ) prepared using the gliotoxin powder.

Cell Culture and Cytotoxicity Assay
The A549 human lung carcinoma epithelial-like cell line was obtained from ATCC (#CCL-185). Cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum Heat Inactivated (FBS) (Gibco Invitrogen, Waltham, MA, USA) and 1% penicillin/streptomycin (P/S) (Gibco Invitrogen, USA). Primary mouse bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from 2-month-old C57BL/6J mice, as described previously [30]. Cells from bone marrow were suspended in PBS and filtered through a 70 µm filter. Isolated cells were cultured in RPMI-1640 medium supplemented with 1% penicillin/streptomycin (P/S) and 12% FBS (Gibco Invitrogen, USA). Non-adherent cells were collected after 24 h by centrifugation and re-cultured in a fresh medium.
For cytotoxicity assay, cells were treated with different concentrations of LH-AuNPs in 96-well plates for 48 h, and an MTT cell proliferation assay kit (Sigma-Aldrich, Darmstadt, Germany) was used to measure the cell viability. Cells were incubated with a medium containing 0.5 mg/mL MTT to metabolize to formazan. Optical density was measured at 550 nm using an ELISA plate reader [31]. Values were expressed as a percentage of control non-treated cells.

Histological Study
The lung was fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained either with hematoxylin and eosin (H & E) or periodic acid-Schiff stained (PAS). Nikon 80i light microscope (Nikon Corporation, Tokyo, Japan) was used to take tissue sections imaged using.

Biochemical Assays
For kidney and liver function assays: serum biochemical markers including aspartate aminotransferase (AST) activity, alanine aminotransferase (ALT), urea, and creatinine were measured according to the instruction manual of commercially available kits from (Abcam, Cambridge, UK).
For antioxidant enzyme measurements: lung tissues were homogenized in HBSS, incubated in ice bath, and centrifuged at 12,000 r/min for 15 min at 4 • C. The supernatants were collected for measurements. Catalase (CAT) activity was determined by measuring the decrease in absorbance of hydrogen peroxide at 240 nm following the method of [32]. Superoxide dismutase (SOD) activity was determined using the adrenochrome test, which relies on the ability of SOD to inhibit the autoxidation of epinephrine in alkaline according to the method [33], and malondialdehyde (MDA) level was measured by the thiobarbituric acid test [34].

Statistical Analysis
All values are expressed as mean ± SD (standard deviation) of at least 3 independent experiments. Power calculation was performed for 2 samples using an unpaired Student's t-test (2-tailed), assuming equal variation in the two groups. Differences were considered statistically significant at * p < 0.05 and ** p < 0.005.

Biosynthesis and Characterization of LH-AuNPs
LH-AuNPs were biosynthesized from an ethanolic leaf extract of L. hastate. The color of L. hastate extract after the addition of aqueous chloroauric acid changed from pale yellow to vivid ruby-red ( Figure 1A). This red color indicated the formation of AuNPs. The biosynthesis of LH-AuNPs was confirmed by UV/Vis spectrum. Different concentrations (1:2 and 1:3) were used for the optimization of AuNPs. Both ratios showed surface plasmon resonance (SPR) peaks in the range of 500-600 nm, which is specific for AuNPs. The most extreme peak with maximum absorbance was recorded at 1:2, which showed an SPR peak at 544 nm ( Figure 2A). TEM revealed a majority of spherical or hexagonal shapes with lattice fringes nanoparticles and sizes ranging from 5 to 30 nm ( Figure 1B). The XRD results 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 ( Figure 2B). of L. hastate extract after the addition of aqueous chloroauric acid changed from pale yellow to vivid ruby-red ( Figure 1A). This red color indicated the formation of AuNPs. The biosynthesis of LH-AuNPs was confirmed by UV/Vis spectrum. Different concentrations (1:2 and 1:3) were used for the optimization of AuNPs. Both ratios showed surface plasmon resonance (SPR) peaks in the range of 500-600 nm, which is specific for AuNPs. The most extreme peak with maximum absorbance was recorded at 1:2, which showed an SPR peak at 544 nm ( Figure 2A). TEM revealed a majority of spherical or hexagonal shapes with lattice fringes nanoparticles and sizes ranging from 5 to 30 nm ( Figure 1B). The XRD results 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 ( Figure 2B).  FTIR spectra of L. hastate extract were expressed in Supplementary Figure S1. Taking the spectrum of plant extract as control, the involvement of different functional groups of L. hastate extract in the reducing and stabilizing process of nanoparticles synthesis was evaluated. Absorbance bands at 3430.5 could be due to the O-H stretching vibration of the phenol groups, which might be responsible for the formation and stabilization of nanoparticles. The peak of 1627.9 cm −1 corresponded to the C=O group. However, the major absorption peak in the FTIR spectra of synthesized AuNPs is related to OH/NH and C=O groups. The presence of OH group could be ascribed to a peak at 3444.6 cm −1 . The peak of 1732.0 cm −1 corresponded to the C=O group ( Figure 2C). The main absorbance band of LH-AuNPs slightly shifted in comparison with the control spectrum. This shifting revealed that biomolecules present in plant extract were responsible for the reduction in gold salt. EDX spectrum of the biosynthesized AuNPs showed strong signals in the gold region that confirmed the formation of AuNPs. A clear, strong peak was observed around 2.40 keV, which is a characteristic of gold nanoparticles ( Figure 2D). There were also some weak signals for carbon and oxygen atoms, which might be due to the X-ray emittance from the enzymes/proteins of the biomolecules involved in the formation and capping of gold nanoparticles. Zeta-potential values are often used as a mark indicative of the stability of colloidal particles. The absolute values replicate the net electrical charge on the particles' external surface that arises from the surface functional groups. Nanoparticles are considered to exist as stable colloids if their zeta potential is more than 25 mV or less than −25 mV [35]. The zeta potential of the LH-AuNPs was −26.1 mV ± 0.2 mV; the suspension of LH-AuNPs in a buffer formed a stable colloid (well-dispersed) with no visible aggregation over 6 months (Supplementary Figure S2). FTIR spectra of L. hastate extract were expressed in Supplementary Figure S1. Taking the spectrum of plant extract as control, the involvement of different functional groups of L. hastate extract in the reducing and stabilizing process of nanoparticles synthesis was evaluated. Absorbance bands at 3430.5 could be due to the O-H stretching vibration of the phenol groups, which might be responsible for the formation and stabilization of nanoparticles. The peak of 1627.9 cm −1 corresponded to the C=O group. However, the major absorption peak in the FTIR spectra of synthesized AuNPs is related to OH/NH and C=O groups. The presence of OH group could be ascribed to a peak at 3444.6 cm −1 . The peak of 1732.0 cm −1 corresponded to the C=O group ( Figure 2C). The main absorbance band of LH-AuNPs slightly shifted in comparison with the control spectrum. This shifting revealed that biomolecules present in plant extract were responsible for the reduction in gold salt. EDX spectrum of the biosynthesized AuNPs showed strong signals in the gold region that confirmed the formation of AuNPs. A clear, strong peak was observed around 2.40 keV, which is a characteristic of gold nanoparticles ( Figure 2D). There were also some weak signals for carbon and oxygen atoms, which might be due to the X-ray emittance

In Vitro Antifungal Activity of LH-AuNPs against A. fumigatus
We examined the antifungal activity of AMB and LH-AuNPs against A. fumigatus using an MTT assay. After 48 h of incubation, the results showed that, with increasing concentrations, the growth of A. fumigatus was significantly inhibited. At concentrations greater than 32 and 64 µg/mL, no fungal colonies were visible in the case of AMB and LH-AuNPs, respectively ( Figure 3A). Thus, we concluded that the MIC of LH-AuNPs was 64 µg/mL. The inhibitory effect of AMB and LH-AuNPs was also observed using inverted microscopy. A strong visual difference in mycelia density and growth was detected among treatments ( Figure 3B). Treatment with 64 µg/mL of LH-AuNPs severely decreased mycelial growth when compared to control. Radial growth of A. fumigatus was repressed by treatment with LH-AuNPs, and 64 µg/mL of LH-AuNPs decreased the relative radial growth by 30% as compared to the control (DMSO). The treated colonies appear to be deficient in green pigmentation, suggesting the formation of little or no conidia ( Figure 3B). Additionally, these phenomena were also detected in liquid culture, where the green pigmentation was completely deficient after incubation with LH-AuNPs ( Figure 3C). The time-kill curves displayed the fungistatic action of both AMB and LH-AuNPs at 32 and 64 µg/mL, respectively, on the growth of A. fumigatus cells ( Figure 3D). After only 8 h of incubation, LH-AuNPs completely inhibited the growth of A. fumigatus to zero colonies ( Figure 3D).

Ultrastructural Analysis of the Interaction between LH-AuNPs and A. fumigatus Cells Using TEM and SEM
The effect of LH-AuNPs (64 µg/mL) on A. fumigatus hypha was studied using SEM. Untreated filaments were smooth and intact with an identical width ( Figure 4A(a,b)). Conversely, variable degrees of cell wall deterioration and damage were observed after treatment with LH-AuNPs. The fungal cell wall exhibited severe pitting, tearing, and penetration in the cytoplasm, with the indication of irregular and rough cell walls, and widespread blebbing. In addition, the hyphae looked distorted, shrunken, and lost structure and rigidity ( Figure 4A(c,d)).
The TEM revealed that untreated hypha of A. fumigatus displayed cells with normal and distinct organelles ( Figure 4B(a,b)). The fungal cell membrane seemed sharp and electron-dense. The outer fibrillary layer showed a lightly distributed, rough, electron-dense layer on the cell wall surface. Conversely, there were prominent alterations in the cell wall, cell membrane, and cytoplasm in LH-AuNPs treated cells ( Figure 4B(c,d)). The inner granular layer of the cell wall was severely disrupted. Aggregates of the outer surface of the cell wall appeared, and a marked disruption of the integrity of the outer cell wall was also ob-

Ultrastructural Analysis of the Interaction between LH-AuNPs and A. fumigatus Cells Using TEM and SEM
The effect of LH-AuNPs (64 µg/mL) on A. fumigatus hypha was studied using SEM. Untreated filaments were smooth and intact with an identical width ( Figure 4A(a,b)). Conversely, variable degrees of cell wall deterioration and damage were observed after treatment with LH-AuNPs. The fungal cell wall exhibited severe pitting, tearing, and penetration in the cytoplasm, with the indication of irregular and rough cell walls, and widespread blebbing. In addition, the hyphae looked distorted, shrunken, and lost structure and rigidity ( Figure 4A(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).

LH-AuNPs Show No In Vitro Cell Toxicity on Animal and Human Cells
We further examined the cytotoxicity of LH-AuNPs on human lung cancer cell line, A549, and primary mBMSCs using cell viability MTT assay. LH-AuNPs showed no cytotoxicity on mBMSCs and human A549 cells up to the concentration of 120 and 150 µg/mL, respectively. A significant reduction in cell viability started to be observed at concentrations of 150 and 200 µg/mL on mBMSCs and human A549 cells, respectively ( Figure 5A). Thus, LH-AuNPs showed very low cytotoxicity on animal cells up to the two folds of the MIC concentration (64 µg/mL). The TEM revealed that untreated hypha of A. fumigatus displayed cells with normal and distinct organelles ( Figure 4B(a,b)). The fungal cell membrane seemed sharp and electron-dense. The outer fibrillary layer showed a lightly distributed, rough, electrondense layer on the cell wall surface. Conversely, there were prominent alterations in the cell wall, cell membrane, and cytoplasm in LH-AuNPs treated cells ( Figure 4B(c,d)). The inner granular layer of the cell wall was severely disrupted. Aggregates of the outer surface of the cell wall appeared, and a marked disruption of the integrity of the outer cell wall was also observed.

LH-AuNPs Show No In Vitro Cell Toxicity on Animal and Human Cells
We further examined the cytotoxicity of LH-AuNPs on human lung cancer cell line, A549, and primary mBMSCs using cell viability MTT assay. LH-AuNPs showed no cytotoxicity on mBMSCs and human A549 cells up to the concentration of 120 and 150 µg/mL, respectively. A significant reduction in cell viability started to be observed at concentrations of 150 and 200 µg/mL on mBMSCs and human A549 cells, respectively ( Figure 5A). Thus, LH-AuNPs showed very low cytotoxicity on animal cells up to the two folds of the MIC concentration (64 µg/mL).

LH-AuNPs Effectively Repair Lung Tissue Damage in IPA Mice without Any In Vivo Toxicity
As shown in Figure 5B, compared to control mice, the IPA mice displayed severe pulmonary lesions described by multifocal infiltrations of macrophages and neutrophils connected with vascular phenomena (necrosis and hemorrhages) and necrosis of alveolar and bronchiolar epithelial cells ( Figure 5B(a)). In addition, a higher number of proliferating hyphae and coagulation necrosis were also detected in the blood vessels, bronchioles, and alveoli, as revealed by the stained section with PAS ( Figure 5B(b)). Interestingly, treatment of IPA mice with LH-AuNPs showed to significantly repair the lung tissue damage, as revealed by less extensive inflammation in H&E staining of lung tissues of LH-AuNPstreated mice (Figure 5B(a)). Moreover, PAS staining confirmed the lack of fungal infection when LH-AuNPs were used ( Figure 5B(a)).
In association with the therapeutic effect of LH-AuNPs in IPA mice, LH-AuNPs did not show any toxicity or damage to the liver or kidney, as mentioned by H&E histological analysis ( Figure 6A). Moreover, biochemical analysis of serum markers for liver function (AST and ALT) and kidney function (Urea and creatinine) were normal and not affected by LH-AuNPs treatment ( Figure 6B).

LH-AuNPs Effectively Repair Lung Tissue Damage in IPA Mice without Any In Vivo Toxicity
As shown in Figure 5B, compared to control mice, the IPA mice displayed severe pulmonary lesions described by multifocal infiltrations of macrophages and neutrophils connected with vascular phenomena (necrosis and hemorrhages) and necrosis of alveolar and bronchiolar epithelial cells ( Figure 5B(a)). In addition, a higher number of proliferating hyphae and coagulation necrosis were also detected in the blood vessels, bronchioles, and alveoli, as revealed by the stained section with PAS ( Figure 5B(b)). Interestingly, treatment of IPA mice with LH-AuNPs showed to significantly repair the lung tissue damage, as revealed by less extensive inflammation in H & E staining of lung tissues of LH-AuNPstreated mice (Figure 5B(a)). Moreover, PAS staining confirmed the lack of fungal infection when LH-AuNPs were used ( Figure 5B(a)).
In association with the therapeutic effect of LH-AuNPs in IPA mice, LH-AuNPs did not show any toxicity or damage to the liver or kidney, as mentioned by H & E histological analysis ( Figure 6A). Moreover, biochemical analysis of serum markers for liver function (AST and ALT) and kidney function (Urea and creatinine) were normal and not affected by LH-AuNPs treatment ( Figure 6B).

LH-AuNPs Excert Significant Reduction of A. fumigatus Colonization and Gliotoxin Production
To measure the efficiency of LH-AuNPs against A. fumigatus infection in IPA mice, we determine the mean fungal burden in the lung of IPA-treated mice against control, IPA-non-treated mice. As shown in Figure 6C, the mean burden of fungal cells in the lung was 96 × 10 6 in LH-AuNPs versus IPA 210 × 10 6 in IPA-non-treated mice ( Figure 6C). In addition, LH-AuNPs significantly reduced the gliotoxin production by A. fumigatus in IPA mice by 69% as compared to the IPA mice ( Figure 6D).

LH-AuNPs Significantly Reduce Inflammation and Oxidative Stress in IPA Mice
As shown in Figure 7A-C, the elevated levels of pro-inflammatory cytokines, including TNFα, IL-1, and IL-17, in IPA mice were significantly reduced in LH-AuNPs-treated mice ( Figure 7A-C). Measurement of antioxidant enzymes activities in the lung of nontreated IPA mice revealed the reduced levels of both CAT and SOD enzymes while increased levels of MDA enzyme ( Figure 7A-C). In contrast, IPA mice treated with LH-AuNPs displayed increased levels of both CAT and SOD and reduced levels of MDA as compared to IPA-non-treated mice group ( Figure 7D-F).

LH-AuNPs Excert Significant Reduction of A. fumigatus Colonization and Gliotoxin Production
To measure the efficiency of LH-AuNPs against A. fumigatus infection in IPA mice, we determine the mean fungal burden in the lung of IPA-treated mice against control, IPA-non-treated mice. As shown in Figure 6C, the mean burden of fungal cells in the lung was 96 × 10 6 in LH-AuNPs versus IPA 210 × 10 6 in IPA-non-treated mice ( Figure 6C). In addition, LH-AuNPs significantly reduced the gliotoxin production by A. fumigatus in IPA mice by 69% as compared to the IPA mice ( Figure 6D).

LH-AuNPs Significantly Reduce Inflammation and Oxidative Stress in IPA Mice
As shown in Figure 7A-C, the elevated levels of pro-inflammatory cytokines, including TNFα, IL-1, and IL-17, in IPA mice were significantly reduced in LH-AuNPs-treated mice ( Figure 7A

Discussion
In this study, we used for the first time the leaf extract of L. hastate to biosynthesize AuNPs with high antifungal activity against A. fumigatus in vitro and in vivo.
We biosynthesized LH-AuNPs using ethanol extract of L. hastate as a green reductant, which improves biocompatibility and pharmacological potential. In consistency with our procedure, it was reported that once the AuNPs are formed in a reaction mixture, the color changes to a ruby red occur because of the surface plasmon resonance (SPR) [36,37]. The absorption peak of the UV spectrum for the biosynthesized LH-AuNPs was 544 nm, which is almost the same absorbance band (at 549 nm) of synthesized AuNPs using extract of Crocus sativus [38].
TEM displayed LH-AuNPs particles about 5-30 nm in size with spherical-, hexagonal-, and triangular-shaped appearances. AuNPs with these distinctive structures have previously been described [39]. In addition, the EDX and XRD analyses verified the presence of AuNPs and their typical metallic gold nano-crystalline structural configurations. It was reported that both the hydroxyl and carbonyl groups are accountable for the stabilization of gold nanoparticles [40]. Our FTIR spectra displayed peaks related to OH and carbonyl groups capped the nanoparticle surfaces. These results are in agreement with reported results of biosynthesized AuNPs using Tetraselmis suesica [41].
In this study, we examined the antifungal activity of LH-AuNPs against A. fumigatus using an MTT assay. Our results showed that the MIC of LH-AuNPs was 64 µg/mL. Green synthesized AuNPs using different plant extracts showed remarkable antifungal activity against Aspergillus species. For example, the results of the serial dilution plate counting

Discussion
In this study, we used for the first time the leaf extract of L. hastate to biosynthesize AuNPs with high antifungal activity against A. fumigatus in vitro and in vivo.
We biosynthesized LH-AuNPs using ethanol extract of L. hastate as a green reductant, which improves biocompatibility and pharmacological potential. In consistency with our procedure, it was reported that once the AuNPs are formed in a reaction mixture, the color changes to a ruby red occur because of the surface plasmon resonance (SPR) [36,37]. The absorption peak of the UV spectrum for the biosynthesized LH-AuNPs was 544 nm, which is almost the same absorbance band (at 549 nm) of synthesized AuNPs using extract of Crocus sativus [38].
TEM displayed LH-AuNPs particles about 5-30 nm in size with spherical-, hexagonal-, and triangular-shaped appearances. AuNPs with these distinctive structures have previously been described [39]. In addition, the EDX and XRD analyses verified the presence of AuNPs and their typical metallic gold nano-crystalline structural configurations. It was reported that both the hydroxyl and carbonyl groups are accountable for the stabilization of gold nanoparticles [40]. Our FTIR spectra displayed peaks related to OH and carbonyl groups capped the nanoparticle surfaces. These results are in agreement with reported results of biosynthesized AuNPs using Tetraselmis suesica [41].
In this study, we examined the antifungal activity of LH-AuNPs against A. fumigatus using an MTT assay. Our results showed that the MIC of LH-AuNPs was 64 µg/mL. Green synthesized AuNPs using different plant extracts showed remarkable antifungal activity against Aspergillus species. For example, the results of the serial dilution plate counting method revealed that the biosynthesized AuNPs using Viola betonicifolia extract significantly inhibited the growth of A. fumigatus, A. flavus, and A. niger with efficacy greater than 67% [42]. The results of the agar well diffusion method showed that biosynthesized AuNPs using Mentha piperita extract were highly active against A. flavus with an inhibition zone diameter (IZD) of 18.49 mm [43]. Similarly, the antifungal potential of licoriceroot-extract-mediated synthesis of AuNPs was very high towards A. flavus with IZD of 18 mm [44].
Our results showed that the LH-AuNPs have moderate antifungal action against A. fumigatus when compared to AMB. Although AMB exhibited high antifungal activity via interacting with sterols of mammalian cells, AMB caused several side effects in treated patients, including higher rates of nephrotoxicity and hypokalemia [45]. Furthermore, only 50% of patients with invasive fungal aspergillosis showed a promising response to AMB treatment, and the survival rates were approximately 59%. On the other hand, AuNPs are recommended for treatment due to their basic antifungal potential or as a drug delivery vehicle with an emphasis on decreasing the dose of antifungal agent required for treatment [46].
The mechanism of the antifungal action of gold ions implicates their absorption and accumulation by the fungal cell, which in turn damages the cell membrane via inhibiting the cell wall β-glucan synthase. Moreover, AuNPs might interact with cell wall macromolecules and membrane proteins [51]. In this context, our SEM and TEM images showed significant morphological alterations in A. fumigatus upon exposure to AuNPs. The ultrastructural modifications include disturbing cell permeability by making pits and gaps in the cell membrane, which leads to structural changes in the outer membrane and fungal cell death. These data are consistent with the reported effects of AuNPs on C. albicans to cause collapse and disintegration of the cytoplasm material [52,53].
In this study, A. fumigatus treated with LH-AuNPs displayed a loss of green pigmentation. Melanin pigments improve the ability of fungi to resist immune clearance, increase virulence and drug resistance, and favor pathogenicity [54]. In A. fumigatus, piomelanins protect hyphal cell walls from ROS and gray-green. DHN-melanins maintain the structural integrity of the cell wall of conidia due to their adhesive properties [55]. In this context, the reduced virulence toward mice was reported for non-melanizing (white) mutants of A. fumigatus [56], while non-pigmented mutant of A. fumigatus was more susceptible to being eliminated by human monocytes as compared to wild-type conidia [57].
Our data were the first to demonstrate the low cytotoxicity of LH-AuNPs on cultured primary BMSCs and MCS5. AuNPs showed different toxicity effects on different cell types; for example, AuNPs induced oxidative-stress-related cytotoxicity in hepatocytes [58] and small airway epithelial cells (SAECs) [59]. On the other hand, Au-NPs caused low long toxicity effect on several other cell lines, including MG63 cells, HeLa cells, and Vero cells [60,61].
We demonstrated the effective treatment of IPA mice with intravenous injection of LH-AuNPs without observing any in vivo cytotoxicity. Many reports investigated the in vivo pharmacokinetic profile of AuNPs after intravenous administration and showed the clearance of Au-NPs from the bloodstream and its accumulation preferentially in the liver, kidney, and spleen. However, these accumulations of AuNPs did not cause any hepatic or renal toxicity [62][63][64]. In consistent, our results demonstrated that LH-AuNPs do not cause any acute or chronic toxicity in vivo.
Production of gliotoxin, an immune-suppressive mycotoxin, is involved in the pathogenesis of IPA and might be a marker of infection with A. fumigatus [65,66]. In this context, our data demonstrated the direct effect of LH-AuNPs administration on inhibiting the A. fumigatus burden by more than 60% and suppressing their gliotoxin production.
The mouse model of IPA demonstrated activation of inflammatory programs by increasing inflammatory cytokine-mediated pathology, including production of IL-1, IL-17, IL-23, and TNF by eosinophils, inflammatory monocytes, dendritic cells (DCs), and alveolar macrophages [67][68][69][70]. Our results demonstrated the effective inhibitory effect of LH-AuNPs on IPA-induced inflammatory cytokines. Similarly, we have recently reported the therapeutic potential of a single dose of green synthesized AS-AgNPs to suppress IPA-induced pro-inflammatory cytokines and lung tissue damage in mice [14]. The antiinflammatory properties of Au-NPs were reported in vitro and in vivo in many studies to be mediated by antioxidant properties [71,72]. AuNPs can act to reduce the production of reactive oxygen and display good free radical scavenging activity against antioxidant enzymes [73]. In addition, the anti-inflammatory properties of Au compounds, which are used for the treatment of inflammatory disorders (such as rheumatoid arthritis, stroke, and cerebral damage), were found to be mediated by inhibiting the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [74][75][76].

Conclusions
The green synthesized LH-AuNPs showed significant antifungal activity against A. fumigatus, including the inhibition of fungal hyphae radial growth and green pigmentation and the deterioration of cell walls. Intravenous administration of LH-AuNPs in the IPA mice model displayed obvious therapeutic potential for lung repair without showing any in vivo cytotoxicity. The therapeutic potential of LH-AuNPs in vivo was found to be mediated via the inhibitory effect of LH-AuNPs on the fungal burden, gliotoxin production, and IPA-induced inflammatory cytokines production in the lung of IPA mice.

Institutional Review Board Statement:
The procedure of the animal study protocol was approved by the Faculty of Science Institutional Animal Care and Use Committee (IACUC), Cairo University, Egypt (CUFS/F/10/13). Data Availability Statement: All materials are available by the corresponding author.

Conflicts of Interest:
The authors declare that they have no competing interest.