Geranium leaf - mediated synthesis of silver nanoparticles and their transcriptomic e ﬀ ects on Candida albicans

: Candida albicans is the most predominant fungal species isolated from medical devices, including catheters, heart valves, and dental prostheses. In recent years, it has been demonstrated to be resistant to many antifungals; therefore, silver nanoparticles ( AgNPs ) have been proposed as an alternative. But only a handful of research is contrib - uted to omic - based studies to study the various impacts of AgNPs on Candida species and other microorganisms. Thus, the study aims to biosynthesize AgNPs using Pelargonium - hortorum leaf and test its antifungal, cytotoxicity, and global gene expression on Candida through transcriptomic pro ﬁ ling. The leaf - assisted AgNPs resulted in spherical shapes with a particle size of 38 nm. The anticandidal e ﬀ ect demonstrated that the Minimum inhibitory concentration was 25 μ g · mL − 1 . Later, the cytotoxicity assay reported a moderate impact on the human gingival ﬁ broblast cells. Finally, the transcriptomic analysis demonstrated the di ﬀ er - ential gene expression of 3,871 upregulated and 3,902 downregulated genes. Thus, proving the anticandidal e ﬀ ect of AgNPs on Candida through RNA - seq experiments and the regulated genes is highly important to cell wall integrity, adherence, and virulence.


Introduction
Candida albicans (C.albicans) is a unique and opportunistic pathogen that frequently dwells in equilibrium with other microorganisms of the commensal mucosal microbiota.However, it is considered a critical resourceful, highly organized yeast causing various forms of candidiasis in immunocompromised patients [1].In the case of a healthy individual, the balance between the host, C. albicans, and the commensal microbiota is maintained.It is due to the complex and dynamic interplay between various immune and environmental factors, such as pH and nutrient availability [2].But the presence of removable dental prostheses, medications like antimicrobials, and behavioral factors such as smoking can cause a variation, affecting the regulatory elements.This may lead to an altered microbial community, which could cause the rapid proliferation of C. albicans, resulting in local and systemic infections [3].
In general, healthy individuals have 20-40% of C. albicans colonization prevalence in the oral cavity, whereas over 60% in immunocompromised subjects, which can pose a severe risk of infection.C. albicans is the most prevalent fungal species isolated from different medical devices, including catheters, pacemakers, heart valves, joint prostheses, contact lenses, and dental prostheses.The continuous mistreatment of C. albicans infection causes antifungal resistance, an emergent problem despite a diverse range of antifungals exploiting different mechanisms of action against fungi.The most frequently used antifungal agents are azoles, polyenes, echinocandins, and nucleoside analogues.Nonetheless, C. albicans biofilms are resistant to most antifungals compared to their planktonic counterparts [4,5].
Azoles, for example, are ineffective against the biofilms of C. albicans [6].To overcome the limitations and drawbacks of traditional antifungal agents, novel antifungals must be developed to combat biofilm-based infections.With the booming development of nanotechnology, versatile nanoscale materials with antimicrobial effects have been designed and exploited against several infections, i.e., nano-antimicrobials (n-AMBs).Nanoparticles (NPs) present a diverse biocidal activity mechanism that is very different from traditional antibiotics.Nanostructured materials have unique physicochemical properties such as their controllable size, large surface area, high reactivity, individual biological interactions, and functional structures [7].Thus, n-AMBs are considered a promising and outstanding alternative in deciphering the problem of microbial resistance [8].In most cases, n-AMBs are in metallic form with a nanometric size that facilitates the internalization of the microorganisms, thereby controlling the proliferation by intervening in the biological mechanisms [9].
The outcome of the n-AMBs area has resulted in an enormous number of metallic NPs effective against several microorganisms [10].One such spectacular NP that is investigated widely is AgNP synthesized by different sources and tested its potential against bacteria, fungi, and viruses [11].These led to the emergence of numerous products in the market for human use.Even though extensive research has been reported, the antimicrobial mechanism of AgNPs is not fully understood.Overall, it is known that Ag + ions bind to proteins and nucleic acids that are negatively charged, causing structural changes and deformations [12].These ions are responsible for forming reactive oxygen species (ROS), primarily affecting the cell membrane through the peroxidation of polyunsaturated phospholipids in a contact-dependent manner in regard to tackling C. albicans and other microorganisms from different origins, various reports have been documented [13,14].However, the essence of AgNPs toxicity still lacks in-depth studies, i.e., on a molecular level.For example, our search found that very few articles represented in Table 1 [15][16][17][18][19][20][21] have made an omic-based analysis in specific transcriptomics.The genetic information encrypted in the cell nucleus is expressed through transcription and translation mechanisms [22].The transcription process depends on the intra/extracellular stimulus that leads to both the expression and repression of genes.Some sophisticated tools have made it possible to study the transcriptomic profile of C. albicans using next-generation sequencing (NGS) technologies, such as RNA-seq [23].The impact of the stimulus's mechanism of action can be studied by analyzing the differentially expressed genes.
Thus, the current research project aims to obtain AgNPs using green technology and evaluate their biological response.Even though every year several studies are conducted based on AgNPs for various biomedical applications, especially antifungal agent against various strains.But many studies have created a void on a effect on cellular/molecular level.Thus, in this research, we have made a comprehensive analysis to fulfill various aspects.Thus, apart from routine testing like cytotoxicity, and antifungal studies, most importantly, gene expression profiling through transcriptomic mediated technique against C. albicans by RNA-sequencing method has been carried out to determine both the up-and downregulation of genes which are affected during the exposure of AgNPs represented in Scheme 1.

Materials and methods
All the chemical reagents were purchased from Sigma-Aldrich TM , Mexico, until otherwise mentioned and used without any further modifications.

AgNPs synthesis
Through chemical synthesis, AgNPs were synthesized using silver nitrate (AgNO 3 , purity ≥99.0%) as a precursor and a filtered Pelargonium-hortorum infusion as a reducing and stabilizing agent.The AgNO 3 precursor solution was prepared at a molar concentration of 25 mM and dissolved in 20 mL of deionized water (DIw).For the preparation of leaf extract, 12 g of Pelargonium tender leaves were weighed, rinsed, and boiled in 100 mL of DIw for 5 min at a temperature of 95°C.Before the synthesis, leaf extract was primarily filtered through 0.2 μm thick Whatman ® filter paper.Initially, 20 mL of ethylene glycol was added to a three-neck flask and heated at 185°C; 10 mL of Pelargonium extract was mixed for 5 min.Subsequently, the AgNO 3 solution was added dropwise every 2 min until a color change was noticed to amber yellow and continued for 90 min to ensure complete reduction of the precursor.Finally, the reaction is allowed to cool down to room temperature and washed twice through centrifugation for 10 min at 4,600 rpm.The pellet is dispersed in a sterile DIw and stored at 4°C until further use for characterization and application studies.

Characterization
The leaf-assisted AgNPs synthesis was confirmed using UV-visible (UV-Vis) spectroscopy (Multiskan GO, Thermo Scientific, Massachusetts, USA), measured in the 200-1,000 nm range.The morphology and size were determined using transmission electron microscopy (TEM, JEOL-1010, JEOL, Massachusetts, USA), where the NP sample was loaded onto a 200 mesh carbon-coated copper grid (Ted Pella, Inc, California, USA).The functional groups of both leaf infusion and synthesized AgNPs were analyzed using Fourier-transform infrared spectroscopy (FTIR, Bruker Tensor-27, California, USA), from 4,000 to 400 cm −1 in a transmission mode with a resolution of 4 cm −1 .The particle size and surface charge were characterized using Zetasizer Nano ZS90 Size Analyzer (Malvern Panalytical, Malvern, UK) using folded capillary cell cuvettes.

Antifungal activity 2.2.1 Candida growth
The antifungal effect of AgNPs was tested using two separate experiments, such as microdilution and colonyforming unit methods.C. albicans ATCC 90028 (Virginia, USA) was cultured aerobically at 37°C on Sabouroad Dextrose Agar (SDA, NutriSelect ® Plus) for 24 h.A single colony was cultured overnight in a Roswell Park Memorial Institute (RPMI) 1640 medium (without glutamine, with red phenol, buffered to pH 7.0 using MOPS).A standard inoculum  [21] 100-125 Escherichia coli K1 Scheme 1: Green synthesis of silver nanoparticles using Pelargonium leaf extract and tested its various biological effects, such as cytotoxicity on fibroblasts cells, anticandidal effect, and finally assessed the global gene expression through transcriptomic profiling on C. albicans.

Microdilution experiment
In a 96-well plate, two-fold serial dilutions of test AgNPs were prepared from 6, 12, 25, 50, 100, and 200 μg•mL −1 .100 µL was added to triplicate wells, followed by an equal volume of test Candida suspension.The RPMI medium (without AgNPs plus Candida) and culture media were used as positive and negative controls, respectively.All the suspensions were then incubated for 24 h at 37°C.Then, the absorbance was measured by OD using a spectrophotometric plate-reader (FLUOstar ® Omega, BMG Labtech, Inc., Bucks, UK).An absorbance reduction of at least 80% compared to positive control was considered to be indicative of Candida growth inhibition.

Colony counting method
After 24 h, 100 µL of the different concentrations tested was added into a sterile tube with 1 mL of PBS.The tubes were vortex for 1 min.Serial dilutions of 9:1 were made and cultured on an SDA-coated petri dish for 24 h, and the colonies were checked to determine the colony growth visually.

Candida morphological analysis
After 24 h, AgNPs untreated and treated samples were obtained and observed under a scanning electron microscope (SEM, Tescan Vega 3, Tescan Ltd, California, USA).They were fixed in 3% glutaraldehyde for 2 h and rinsed thrice.The dehydration process was made using 50%, 70%, 90%, and 100% ethanol concentration series.Then, hexamethyldisilazane treatment was added to the samples and kept on a fume hood overnight.Finally, the samples were sputter coated (K650x sputter coater, Quorum Technologies, Lewes, UK) with gold and analyzed in the microscope.

MTT assay on HGF cells
The reduction of the bromide salt of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazole (MTT) test was used to determine the cytotoxic activity of AgNPs on human gingival fibroblasts-1 (HGF-1) cell line ATCC CRL2014 and primary culture (HGF).All cell culture reagents mentioned below were bought from Gibco™, Thermo Fisher Scientific, USA.The cell density equivalent to 1 × 10 5 cells•mL −1 was placed in a 96-well plate (100 µL) in Dulbecco's modified eagle medium (DMEM), which was previously supplemented with 10% fetal bovine serum, 1% glutamine (Glutamax), and 2% of antibiotics.The cells were incubated for 48 h at 5% CO 2 at 37°C.Then, serial dilutions of AgNPs (0-1.62 μg•mL −1 ) were inoculated and incubated for 24 h under the same conditions.After 24 h, the medium was removed, and the freshly prepared MTT bromide salt at a concentration of 0.2 mg•mL −1 in supplemented DMEM was added to each well.The 96-well plate was incubated for 4 h, then, the formazan crystals were dissolved using dimethyl sulfoxide (DMSO, Karal, León, Mexico), and readings were analyzed using a Multiskan GO spectrophotometer at 570 nm.The experiment was performed in triplicates from three samples.

Transcriptomic expression profile
Total RNA extraction was performed in C. albicans with and without AgNPs (20 µg•mL −1 ) treatment incubated for 24 h at 37°C using the RiboPure Yeast Kit (Invitrogen™, Massachusetts, USA) by following the manufacturer's protocol.From a 24 h grown culture in SDA, three colonies were placed in three flasks with 5 mL of SDA.Subsequently, they were incubated at 37°C under stirring at 170 rpm for 24 h.Two groups were prepared: (1) C. albicans control group and (2) C. albicans experimental group (AgNPs treated).Both the quality and quantity of RNA were estimated by measuring the absorbance at 260 and 280 nm by UV spectroscopy and using NanoDrop2000™ (Thermo Fisher Scientific Inc, USA) by placing 1 µL of each sample.

RNA-seq analysis
To explore the impact of the AgNPs exposure on C. albicans, we performed RNA-seq using NextSeq 500 system (Illumina, Inc, California, USA), where 2 × 75 cycles pairend readings were conducted, and 60 million reads were obtained.The process is as follows: 2 µg of extracted RNA was dispersed in 50 μL of RNase-free water (Invitrogen™, Massachusetts, USA), and the analysis was carried out on the system.The samples were sequenced in triplicates; the generated reads were mapped to the Candida 5,314 genome.The statistical analysis of differentially expressed genes was performed using edgeR software with a two-fold change and a P-value of <0.01.

Synthesis and characterization
To confirm the synthesis of AgNPs, UV-Vis spectroscopy measurements (Figure 1) were carried out that indicate the presence of two distinct peaks.The peak is at 275 nm, and another maximum absorbance is at 420 nmalso the color changes of the precursor solution change to amber yellow.Then the morphology was studied using TEM (Figure 2a), representing a nearly spherical structure.The histogram in Figure 2b depicts the average particle size distribution calculated using ImageJ software and results in a size range of 30-50 nm.We also determined the hydrodynamic diameter and zeta potential of AgNPs shown in Figure 2c and d.The data indicate that the diameter was seen in a bimodal distribution of 48.77 and 176.4 nm.The surface charge was found to be −12.3mV.
The functional groups of both the leaf extract and the as-synthesized AgNPs were obtained by FTIR spectrum, which is shown in Figure 3, which depicts the bands located at 1,387, 1,630, 2,184, and 3,418 cm −1 in both the cases with a minor shift in point of AgNPs while comparing with the extract spectra.

Antifungal activity against C. albicans
The antifungal activity for AgNPs with different concentrations against C. albicans is represented in Figures 4 and 5.
From the microdilution experiment, we found that the minimum inhibitory concentration (MIC) was 25 μg•mL −1 , and the subsequent concentration inhibited the fungal growth to its maximum.While in colony counting, the concentrations range from 6-25 μg•mL −1 , where C. albicans growth is seen.And interestingly, no colonies were identified for concentrations such as 50-200 μg•mL −1 .The control positive was saturated with fungal growth, which becomes uncountable compared to treated samples.Based on the effect, SEM observations are shown in Figure 6 to determine the morphology of the C. albicans treated with AgNPs (25 μg•mL −1 ).Indeed, there are morphological changes, resulting in deformations and irregularity of membrane (indicated with red arrows) compared to the control group, which looks smooth and with a stable cell wall surface.

Transcriptomic expression profile of C. albicans
Raw sequencing reads were filtered to remove low-quality reads using trimmomatic before subsequent analysis.Three biologically independent samples were analyzed for each condition by RNA-seq.The control group (without AgNPs treatment) obtained 28,503,316 reads for the experiment (92.88% of total reads).For the experimental group (AgNPs treatment), 30,430,674 reads were obtained (91.57% of total reads).The data were mapped to C. albicans SC5314.The biological replicates were very close, as shown in Figure 8a.The volcano plot indicates upregulated and downregulated genes under the two conditions; each dot represents an individual gene's statistical significance (P-value) versus the magnitude of change (fold-change).Most upregulated genes are toward the right all of which are involved in Biological impact of green nano-silver on C. albicans gene expression  5 ergosterol and diacylglycerol biosynthesis.The most downregulated genes are on the left, with regard to C. albicans adherence and virulence genes (Figure 8c).
Heatmap from Figure 8c shows the hierarchical clustering of the 500 most differentially expressed genes reported by edgeR analysis according to fold-change.Red indicates higher gene expression levels, while beige indicates lower expression by reads per kilobase of transcript per million reads mapped (RPKM) in both conditions.Heatmap (Figure 8c) shows hierarchical clustering and the 500 most variable expressed genes between both conditions.RNA-seq results revealed that many genes in C. albicans were differentially expressed after AgNPs treatment.Gene expression values were quantified as RPKM, where a total of 3,902 genes were downregulated, and 3,891 genes were upregulated.Based on the search for differential expressions on genes widely reported in the literature, we found that these genes are essential for developing C. albicans biofilm formation, adhesion, pathogenicity, and virulence represented in Table 2.

Discussion
AgNPs and their function as an antimicrobial application have become indispensable in n-AMBs resulting in various forms to tackle different kinds of Candida species [24,25].More research is carried out every day to treat C. albicans infections, as it is one of the life-threatening microorganisms.Multiple studies have been published in the past 5 years regarding treating C. albicans with AgNPs, as shown in Table 3 (some examples are listed).A common practice of AgNPs synthesis is exploiting different natural constituents to decrease the toxic effect and have synergistic mechanisms.Even though the results are promising broadly but lack the concept of explaining on a cellular level; thus, this study is purposely dedicated to identifying the effect of AgNPs on the global gene expression of C. albicans through transcriptomic analyses.UV-Vis spectroscopy shows the absorbance of a pure extract with an absorbance of 277 nm, which corresponds to polyphenols [26].These components are secondary metabolites of diverse plants resulting from a reaction to stress stimulus.Various plant extracts can reduce Ag + ion to Ag 0 due to poly hydroxyl and carboxyl groups present in these metabolites [27], Whereas the spectra for synthesized AgNPs resulted in absorbance of 410 nm, confirming the formation of NPs whose characteristic color is amber yellow [28,29].The spectral range from 400 to 420 nm corresponds to spherical AgNPs [30], inferring a particle size between 35 and 50 nm, as reported in the literature.Also, from the spectra, the extract's intensity has been diminished to maximum, demonstrating that this group of molecules is responsible for the process of reducing AgNPs [31,32].
From the morphological analysis using TEM, nearly spherical-shaped and uniformly distributed AgNPs were found with minor organic content of the extract, which helps stabilize the NPs and avoid aggregation or clustering.The size was measured using the histogram and was found to be 30-44 nm with an average particle size of 38 nm [33,34] using ImageJ software by considering 302 particles.The analysis of hydrodynamic diameters (HDD) and zeta potential (ZP) plays a vital role in determining the interaction of NPs in biological entities.Thus, we analyzed HDD for synthesized AgNPs, resulting in dual modal particle size distribution.It is due to the medium in which the NPs are dispersed.Thus, the size is more significant when compared to TEM analysis as it is visualized in a dry state.Also, the Brownian movement significantly impacts determining NPs size when dispersed in the liquid medium.Apart from this, various biological compounds in the extract, such as proteins attachment through amino groups or cysteine residues, participated in the activity of both reducing and stabilizing agents [29,35].The TEM analysis corroborates the obtained results [36].The negative ZP value was determined in the case of obtained AgNPs, and the negative surface might be due to biomolecules present in the leaf extract [37].Also, the ZP explains that the AgNPs are aggregated minorly, similar to the other reported literature using plant extracts [38].
The plant extracts are usually made of various organic reducing agents such as phenolic compounds, terpenes, polysaccharides, etc. [31,32].So, FTIR characterization is one of the primary methods to analyze different functional groups.Thus, we employed this method to study the groups of leaf extract and AgNPs.The results show that both samples showed similar bands of flavonoids and terpenoids present in the leaves that highlight the presence of residues from the Pelargonium infusionthese  compounds aid in stabilizing the AgNPs by attaching to the NPs on the surface [39].This confirms that the extract's organic components are involved in reducing Ag metal.From both spectra, we found various groups with the corresponding bands ate 3,310, 2,122, 1,634, 1,385, 1,334, and 1,040 cm −1 [40].The strong and sharp band at 3,310 and 1,634 cm −1 corresponds to alcoholic O-H and N-H stretching, respectively.The weak band at 2,122, 1,385, 1,334, and 1,040 cm −1 are assigned to the C]N, C-O-H bending vibration (phenolic group), -C-O stretching, and C-O stretching vibration of the OH group, which reveals the presence of phenolic compounds in the extract.In the case of AgNPs, almost similar bands are visualized but with minor shifts like 2-4 cm −1 , confirming that the various functional groups have extensively interacted with Ag + ions [32,39].
AgNPs antifungal efficiency depends on parameters like shape, size, and surface charge [41].The smaller-sized AgNPs with spherical forms can have the maximum capacity to release Ag + ions due to the larger surface area.The MIC obtained in the present study was 25 μg•mL −1 , similar to the reported in the literature [42] and less than the reports published with the ATCC90028 strain [43].AgNPs' serial dilutions were tested on C. albicans in SDA agar plates, and after 24 h of incubation, it showed no growth.The concentrations of >25 μg•mL −1 of AgNPs tested were effective as there is no fungal growth, and they did not recover after treatment.Thus, in our study, the particle size plays a vital role in determining its antifungal effect by binding ions to -OH groups and internalizing through the cellular membranes leading to exposed atoms and available for redox reactions and high accumulation of ROS causing damage to nucleic acid leading to apoptosis [25,44,45].
SEM imaging shows a deformed and irregular cell wall when treated with AgNPs.It has been reported that AgNPs can effectively disrupt cell walls creating pits [46,47].This damage plays an essential role in interaction and adhesion to the host tissue, which is crucial for the first stages of C. albicans invasion [48].
HGF and HGF-ATCC were tested for the cytotoxic effect of AgNPs, demonstrating that HGF ATCC was more susceptible to AgNPs than HGF, as reported [49].It is well known that AgNPs have a cytotoxic effect on several 5human cells [50,51] in a dose-dependent manner, as reported in the present study.Concentrations ranging from 0.0015 to 0.006 μg•mL −1 exhibited an hormesis effect.In contrast, very low concentrations stimulate cell proliferation interestingly.Some research works have reported that it is due to the activation of the nuclear factor erythroid-derived two related factor 2 (Nrf2) [52].Pathways of MAPK are involved in the regulation of cell proliferation and the regulation of catabolic pathways during cell stress that translates cell growth, differentiation, and apoptosis [53].
The most expressed gene in C. albicans without NPs treatment is the WH11 gene.It is well known that C. albicans can switch from white to opaque states.This occurs spontaneously and implicates phenotypic changes in cell wall morphology, size, adhesion to host, and drug susceptibility/resistance [54].The white states confer the ability to be more virulent than opaque states, which are preferable for rapid multiplication to form biofilm structures [55].
The most expressed genes in C. albicans with NPs treatment were more concerning the synthesis of cell wall components such as diacylglycerol (PLD1) that encodes phospholipase, a protein implicated in the change from yeast to hyphae [56].PHR1 has transferase activity of beta-(1,3)-glucanosyltransferases that is fundamental for cell wall structure.Studies have reported when deletion of this gene in C. albicans confers less capacity to adherence to surfaces and epithelial cells [57].ERG3 chains that it is a protein that catalyzes the induction of C-5 double bond that contributes to the biosynthesis of ergosterol, an essential component of the cell wall [58].It should be noted that the sequencing data confirm what was observed in SEM microscopy, where changes in the cell wall were observed in response to oxidative stress; we can confirm that the AgNPs have an oxidation mechanism in Candida, as they have also been previously reported.CDR2 encodes a multidrug output transporter that, compared to the genes that were expressed in Candida when they were not in contact with the NPs, it is observed that in this condition, it was not expressed, so we conclude that AgNPs caused a toxicity effect that Candida recognized as a threat so that it activates the mechanisms similar to those that it activates when in contact with antifungals [59].
In contrast, genes that were less expressed or downregulated are ALS1 and ALS3, a protein necessary for surface adhesion and host invasion [60,61].Finally, another important family of proteins was notably downregulated: SAP family proteins, such as SAP 4 and 6, are essential as they involve virulence and tissue penetration; it degrades the keratin found in the soft tissues leading to invasion [62].Several levels of expression of C. albicans genes treated with AgNPs are responsible for reducing its effect on the host interaction as a consequence of suppression.Previous studies have reported a change in expression levels of genes associated with cell virulence, adherence, and biofilm formation [63].

Conclusions
In the current scenario, many investigations have explored the various biomedical applications of AgNPs and their development as effective antifungal agents.Most studies lack in-depth knowledge on the omic level to elucidate the antimicrobial function.The synthesis of AgNPs assisted with Pelargonium leaf extract showed that formed NPs are spherical morphology with an approximate size of 38 nm and high stability.AgNPs showed antifungal effectiveness as a possible solution to the problem of resistance to various therapeutic agents.The overall results from omic profiling show that the expression of genes is upregulated and downregulated, which is of great importance to the virulence, adhesion, and biological activity of C. albicans by treating with AgNPs.All of those, as mentioned earlier, suggest a vital role in these genes' cellular response to AgNPs.However, more studies need to be carried out to make the AgNPs with the possible application in biomedicine, especially n-AMBs.

Figure 7
Figure7corresponds to the MTT assay, and the graph compares the cytotoxicity effect of AgNPs on the two HGF cells.Both cases show a cytotoxic effect in a dosedependent manner.But from the graph, we found that HGF no CC 50 and HGF-ATCC was 1.05 μg•mL −1 , and a hormesis effect was observed with almost all the concentrations of more than 50% cell viability.

Figure 1 :
Figure 1: Represents the optical study of Pelargonium extract and synthesized AgNPs using UV-Vis spectroscopy (inset: shows the color of AgNPs after being reduced by Pelargonium extract).

Figure 2 :
Figure 2: (a) AgNPs morphological study using TEM, which is spherical.(b) Histogram of particle size distribution calculated using ImageJ.(c) Hydrodynamic diameter of AgNPs shown in a bimodal distribution.(d) Zeta potential of as-synthesized NPs.

Figure 3 :
Figure 3: FTIR characterization of the Pelargonium extract and synthesized AgNPs indicating the various functional groups.

Figure 4 :
Figure 4: Antifungal studies using the microdilution method, and the graph shows the dose-dependent effect (n = 36).

Figure 5 :
Figure 5: Shows the photos of Petri plates used for colony counting studies.

Figure 6 :
Figure 6: Morphological assessment of C. albicans: (a) control and (b) AgNPs treated and their effect on the fungal structure (marked in red arrows).

Figure 7 :
Figure 7: Cytotoxicity evaluation of HGF primary cells and HGF ATCC cell line using MTT after incubating AgNPs for 24 h (n = 36).

Figure 8 :
Figure 8: Transcriptome analysis of C. albicans by RNA-seq.(a) Multidimensional scaling plot, which determines the most significant data variation sources.(b) Volcano plots that correspond to the differentially expressed genes.Upregulated genes (red dots-right), downregulated genes (red dots-left), no significant differential expressed genes (black dots).(c) Heat map of 500 differential gene expressions between the control group (C.albicans without AgNPs treatment) and experimental group (C.albicans treated at 25 µg•mL −1 ).

Table 1 :
Investigations that studied the impact of AgNPs against different microorganisms through transcriptomic profiling

Table 2 :
Genes of interest, differential expression gene is represented in fragments per kilo base of transcript per million mapped fragments (FPKM) comparing control group vs experimental group

Table 3 :
Green synthesized AgNPs against different C. albicans strains