Functional Attributes of Myco-Synthesized Silver Nanoparticles from Endophytic Fungi: A New Implication in Biomedical Applications

Simple Summary Greener fabrication of silver nanoparticles has received extensive attention owing to their low cytotoxicity, tunable surface features, and stability. Fabrication of AgNPs using natural entities has received substantial consideration as an alternative to toxic chemicals. In this study, we have screened the potential of an endophytic fungi Penicillium oxalicum strain LA-1 for the synthesis of AgNPs and analyzed their potential multipurpose values in biology and medicine. Abstract To develop a benign nanomaterial from biogenic sources, we have attempted to formulate and fabricate silver nanoparticles synthesized from the culture filtrate of an endophytic fungus Penicillium oxalicum strain LA-1 (PoAgNPs). The synthesized PoAgNPs were exclusively characterized through UV–vis absorption spectroscopy, Fourier Transform Infra-Red spectroscopy (FT-IR), X-ray powder diffraction (XRD), and Transmission Electron Microscopy (TEM) with energy dispersive X-ray spectroscopy (EDX). The synthesized nanoparticles showed strong absorbance around 430 nm with surface plasmon resonance (SPR) and exhibited a face-centered cubic crystalline nature in XRD analysis. Proteins presented in the culture filtrate acted as reducing, capping, and stabilization agents to form PoAgNPs. TEM analysis revealed the generation of polydispersed spherical PoAgNPs with an average size of 52.26 nm. The PoAgNPs showed excellent antibacterial activity against bacterial pathogens. The PoAgNPs induced a dose-dependent cytotoxic activity against human adenocarcinoma breast cancer cell lines (MDA-MB-231), and apoptotic morphological changes were observed by dual staining. Additionally, PoAgNPs demonstrated better larvicidal activity against the larvae of Culex quinquefasciatus. Moreover, the hemolytic test indicated that the as-synthesized PoAgNPs are a safe and biocompatible nanomaterial with versatile bio-applications.


Introduction
Innovative materials with superior optical, physical, and electronic properties will always find a large spectrum of applications in physics, chemistry, and the biological

Chemicals and Reagents
All the chemicals and reagents used in this study were of analytical grade, purchased from Sigma-Aldrich (St. Louis, MO, USA) and Himedia (Mumbai, India).

Isolation and Characterization of Endophytic Fungi
Our previous report provided a detailed methodology for the isolation and characterization of the endophytic fungi P. oxalicum LA-1 strain from the medicinal plant Limonia acidissima. The isolated strain was sequenced using the internal transcribed spacer (ITS) region and submitted in GenBank with the accession no: KX622790 [20].

Preparation and Extraction of Culture Filtrate from P. oxalicum LA-1 Strain
The fungal biomass was prepared by growing fungi aerobically in potato dextrose broth (PDB) and was incubated at 24 ± 1 • C for 1-7 days. After incubation, the fungal mat was washed twice with double-distilled water, transferred to a flask containing 100 mL of sterile distilled water, and kept under an orbital shaker at 140 rpm for 48 h at 24 ± 1 • C. After 48 h, the culture filtrate was filtered through Whatman filter paper no. 1, and the extract was stored for further use.

Natural Products Analysis by GC-MS
GC-MS detected the active constituents present in the LA-1 strain culture extract. For the analysis, an Agilent GCMS apparatus, GC: 7890A, MSD5975C-HP-5 capillary column (30 m-0.25 mM, ID, a film thickness of 0.25 mM) coupled directly with single quadrupole-MS was used. Furthermore, the mass spectrum interpretation was conducted using the National Institute and Standard Technology (NIST) library. The compound name and molecular weight of the obtained spectrum were identified.

Fabrication of Silver Nanoparticles Using Culture Filtrate of P. oxalicum LA-1 Strain (PoAgNPs)
For the fabrication of PoAgNPs, aqueous filtrate of P. oxalicum LA-1 strain was challenged with aqueous silver nitrate solution. Briefly, for the synthesis of PoAgNPs 10 mL of the filtrate was mixed with 90 mL of 1 mM AgNO 3 and the mixture was incubated at room temperature for 1 to 3 days.

Characterization of PoAgNPs
After the incubation, a change in the reaction's color formation implied the preliminary confirmation of PoAgNPs. To characterize the PoAgNPs, sample preparation and processing were carried out according to our previously established methodology [21]. UV-vis spectrophotometry was used to measure the surface plasmon resonance (SPR) of PoAgNPs, which was operated at wavelengths of 300-700 nm using JASCO V-650 UVvisible spectrophotometers. The plausible functional groups present in the filtrate may act to reduce, cap, and stabilize PoAgNPs. This was ascertained by employing Fourier transform infrared spectroscopy analysis using a Jasco Fourier transform infrared spectrometer performed in the range from 500-3500 cm −1 . X-ray diffraction analysis (XRD) analysis was carried out to probe the crystallographic structure and orientation of PoAgNPs using the Ultima IV X-ray powder diffractometer with CuKα radiation (λ = 0.1546 nm) (Rigaku Ltd., Tokyo, Japan). Transmission electron microscopy (TEM) exclusively characterized the morphology features (size and shape) of PoAgNPs using a JEOL 3010 transmission electron microscope. Zeta potential measurements evaluated the surface charge of the synthesized PoAgNPs by using a Zetasizer Nano (ZS 90) instrument. For DLS analysis, synthesized PoAgNPs were sonicated for 10-20 min to disperse the nanoparticles and were analyzed using the Malvern Zetasizer Nano-ZS90 analyzer.

Hemolytic Activity of PoAgNPs
In a sterile lithium heparin container, blood was collected from healthy volunteers. Red blood cells (RBCs) were then separated using Ficoll density gradient centrifugation (1500× g rpm for 10 min at 4 • C). Next, the RBCs were diluted in 20 mM HEPES buffer saline (pH 7.4) to produce a 5% v/v solution. One mL of diluted PBS was transferred to Eppendorf tubes containing 1% Triton X-100 (positive control), RBC saline (negative control), and different concentrations of PoAgNPs (25,50,75, and 100 µg/mL), which were then added to the tubes; then, the samples were incubated. The samples were centrifuged at 12,000× g RPM at 4 • C, and the supernatant was transferred to 96 well plates. The absorbance was measured at 570 nm [22].
The percent of hemolysis was calculated as follows: × 100

Antibacterial Activity of PoAgNPs
The antibacterial activity of PoAgNPs was evaluated against human bacterial pathogens. The Gram-negative stains Klebsiella pneumonia (MTCC-530), Vibrio cholerae (MTCC 3906) and Escherichia coli (MTCC-1687), and the Gram-positive strains were Micrococcus luteus (MTCC 1809), Mycobacterium smegmatis (MTCC-994) and Bacillus subtilis (MTCC-2387) were used. For the study, bacterial cultures were inoculated and grown overnight. A 6-mm diameter well was made in Mueller-Hinton agar (MHA) media plates using a borer cork. After the incubation hour, the bacterial cultures were spread uniformly throughout the media plate by using a cotton swab. Different concentrations of PoAgNPs (10 µg/mL, 20 µg/mL, 30 µg/mL, and 40 µg/mL) were added to the MHA plates and allowed to stand for one hour for the perfusion of PoAgNPs into the medium. Finally, the petri plates were incubated at 37 • C for 24 h. The zone of inhibition (ZOI) was measured around the wells in diameter (mm) using a meter ruler. The assay was carried out in triplicates with antibiotic streptomycin as a control [23]. The minimum inhibitory concentration (MIC) was performed in 96 well-rounded bottom microtiter plates using the microdilution method [24]. The bacterial strains were cultured in Mueller-Hinton agar and the turbidity was adjusted to a 0.5 McFarland Standard for analyze the MIC. Briefly, One hundred µL of sterile Mueller-Hinton broth was added to the 96-well plate. In the plate, the first two rows were served as a growth control. For analysis, PoAgNPs were serially diluted in dimethyl sulfoxide (DMSO) to create a concentration sequence from 100 to 3.12 µg/mL and added to the wells having previously inoculated bacterial cultures. Finally, 10 µL of Resazurin dye was added to these wells and incubated for 24 h at 37 • C.
The MBC values were defined as the least concentration of antimicrobial agents that prevent the growth of the organisms. The MBC test was performed by plating the suspension from each well of the 96-well plates in sterilized MHA plates followed by incubation for 24 h.

Fluorescence-Based Study of the Bactericidal Activity of PoAgNPs
A log phase culture of bacterial pathogens K. pneumonia, V. cholerae, E. coli, M. smegmatis, M. luteus, and B. subtilis was centrifuged at 4 • C, 10,000× g RPM for 5 min and suspended in phosphate buffer saline (PBS). The supernatant was discarded, and the remaining bacterial cells were resuspended in 5 mL of PBS. Next, 100 µL of 100 µg mL −1 PoAgNPs were added to the bacterial suspensions, incubated for 1 h, and stained using 100 µL of fluorescent dyes (AO/EB) for 15 min. After rinsing the stained sample with PBS, Biology 2021, 10, 473 5 of 20 the images were observed using an OLYMPUS DP27 fluorescence microscope. The control assay was performed without any PoAgNPs treatment.
2.9. In Vitro Anticancer Activity 2.9.1. Cell Lines The human adenocarcinoma breast cancer cell line (MDA-MB-231) was obtained from the National Center for Cell Science (NCCS), Pune, India. The cells were maintained in a DMEM medium supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA) and with 100 µg/mL streptomycin as an antibiotic (Himedia) at 37 • C and 5% CO 2 atmosphere in a CO 2 incubator (EC 160, Nüve, Ankara, Turkey).

MTT Assay
The viability of PoAgNPs-treated MDA-MB-231 cells was measured using the MTT assay [25]. For this, the human adenocarcinoma breast cancer cells (MDA-MB-231) at 1.5 × 10 6 cells/well were seeded in 96-well plates to treat with different concentrations of PoAgNPs (10-100 µg/mL) and incubated in a CO 2 incubator for 24 h. After incubation, 20 µL of MTT solution (5 mg/mL in phosphate-buffered saline) was added. After 4 h, the purple formazan product was dissolved by adding 100 µL of 100% DMSO to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm using a 96-well plate reader (Bio-Rad, Hercules, CA, USA). All the assays were done in triplicate, and the results were given in mean ± SD The cell suspension of the control and the PoAgNPs-treated cells containing 5 × 10 5 cells were added with 20 µL of Acridine Orange (AO) and Ethidium Bromide (EB) solution (3.8 µM of AO and 2.5 µM of EB in PBS) and examined with a fluorescence microscope (Olympus, Japan) using a UV filter (450-490 nm). The staining declared the cells, the nuclear morphology, and the membrane integrity; morphological changes were also observed and photographed [25].

Larvicidal Activity of PoAgNPs
The methodology was adopted from our previous report [26]. Briefly, different concentrations of PoAgNPs (0.31, 0.62, 1.25, 2.5, 5, and 10 ppm) were prepared. Twenty-five numbers of II and IV larval stages of C. quinquefasciatus were introduced into the beaker containing 249 mL of water and 1 mL of above mentioned concentrations of PoAgNPs and kept in an environmental chamber at 25 • C with a 16:8 light/dark cycle. Mortality was noticed after 24 h post-exposure. During the experiment, no food was provided to the larvae. We performed three replications to validate the results. The control (water) was maintained to assess the natural mortality of the mosquito larvae within the test period.
The percentage of mortality was calculated by using the formula: Percentage mortality (%) = ((X − Y)/X × 100) X-the number of live larvae introduced; Y-the number of live larvae treated.

Statistical Analysis
Experiments were carried out using a completely randomized method, and the results were shown as mean ± SD (standard deviation). The SPSS software package 16.0 edition was used for statistical analysis (SPSS Inc., Chicago, IL, USA). One-way ANOVA (post-hoc (Tukey's test)) was performed to know the statistical difference in antibacterial studies. On the other hand, the larval mortality data was subjected to Probit analysis to calculate LC 50 , LC 90 , the 95% confidence interval (upper confidence limit) and the lower confidence limit, the Chi-squares intercept, the Chi-square test, the linear regression, and the F-and R-values.

Results
3.1. Biosynthesis of Silver Nanoparticles from Endophytic Fungi P. oxalicum Strain LA-1 (PoAgNPs) In this study, the biosynthesis and the optimization of silver nanoparticles from extracts of the endophytic fungi P. oxalicum strain LA-1 ( Figure 1) isolated from the medicinal plant L. acidissima were carried out.

Statistical Analysis
Experiments were carried out using a completely randomized method, and the results were shown as mean ± SD (standard deviation). The SPSS software package 16.0 edition was used for statistical analysis (SPSS Inc., Chicago, IL, USA). One-way ANOVA (post-hoc (Tukey's test)) was performed to know the statistical difference in antibacterial studies. On the other hand, the larval mortality data was subjected to Probit analysis to calculate LC50, LC90, the 95% confidence interval (upper confidence limit) and the lower confidence limit, the Chi-squares intercept, the Chi-square test, the linear regression, and the F-and R-values.

Biosynthesis of Silver Nanoparticles from Endophytic Fungi P. oxalicum Strain LA-1 (PoAgNPs)
In this study, the biosynthesis and the optimization of silver nanoparticles from extracts of the endophytic fungi P. oxalicum strain LA-1 ( Figure 1) isolated from the medicinal plant L. acidissima were carried out.

GC-MS Analysis of Bioactive Compounds from Endophytic Fungi
The bioactive compounds present in the LA-1 culture filtrate were examined by GC-MS analysis. Based on the elution order, in the HP-5MS column, the compounds were identified and characterized. In Figure 2, the spectrum for LA-1 extract is provided. We annotated the spectrum with the NIST database and identified 28 compounds present in the extract, as represented in Table 1. Among the identified compounds, four compounds possessed the potential pharmacological activities (pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl); E-14-hexadecenal; 2,5-piperazinedione, 3,6-bis(2-methyl propyl)-and n-hexadecanoic acid) ( Figure 2).

GC-MS Analysis of Bioactive Compounds from Endophytic Fungi
The bioactive compounds present in the LA-1 culture filtrate were examined by GC-MS analysis. Based on the elution order, in the HP-5MS column, the compounds were identified and characterized. In Figure 2, the spectrum for LA-1 extract is provided. We annotated the spectrum with the NIST database and identified 28 compounds present in the extract, as represented in Table 1. Among the identified compounds, four compounds possessed the potential pharmacological activities (pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl); E-14-hexadecenal; 2,5-piperazinedione, 3,6-bis(2-methyl propyl)-and n-hexadecanoic acid) ( Figure 2).

UV Vis Spectroscopy Analysis
When aqueous culture filtrate is mixed with silver nitrate solution, the prompt reaction is initiated. The reaction gradually proceeds with a color change from white to a dark brown color within 24 h at 37 • C. The optical property of PoAgNPs is displayed in Figure 3a-d. To achieve stable and polydispersed PoAgNPs, we optimized various parameters by regulating the combination of silver nitrate salt and LA-1 supernatant, the stoichiometric proportion, the temperature, and the pH (Figure 3a-c). In Figure 3b, the pH 9 absorbance shift increased, indicating an increase in the synthesis of PoAgNPs. The alkaline pH will favor the reaction by an upsurge in the active constituents present in the extract. Temperature plays a crucial role in nanoparticle synthesis. In the present study, synthesis at 37 • C showed the maximum absorbance and a greater yield of nanoparticles (Figure 3c). The optimized synthesis parameters of quantity of culture extract (10 mL), concentration of silver nitrate (1 mM) and stoichiometric proportion of fungal extract: aqueous silver nitrate (10:90 mL), reaction temperature 37 • C, pH 9, incubation time of 24 h produced stable and polydispersed PoAgNPs.

XRD Analysis of PoAgNPs
The crystalline nature of PoAgNPs was ascertained by using XRD analysis. The XRD spectrum for PoAgNPs is depicted in Figure 4b. From the XRD spectrum, four diffraction peaks were revealed at 27.71°, 32.20°, 46.21°, and 54.78°. These can be indexed as (111), (200), (220), and (311), which reflect the face-centered cubic crystalline nature of PoAgNPs.

TEM and EDAX Analysis of PoAgNPs
TEM analysis revealed that PoAgNPs particles were randomly distributed with varying sizes and shapes (Figure 5a-d). Moreover, based on the TEM images' analysis, the mean particle size of PoAgNPs was found to be 52.26 nm (Figure 5e). Elemental analysis of PoAgNPs was performed by EDAX, which displayed Ag as a major element present in the PoAgNPs (Figure 5f).

TEM and EDAX Analysis of PoAgNPs
TEM analysis revealed that PoAgNPs particles were randomly distributed with varying sizes and shapes (Figure 5a-d). Moreover, based on the TEM images' analysis, the mean particle size of PoAgNPs was found to be 52.26 nm (Figure 5e). Elemental analysis of PoAgNPs was performed by EDAX, which displayed Ag as a major element present in the PoAgNPs (Figure 5f).

Zeta Potential Analysis and DLS of PoAgNPs
Surface charge and stability are the essential phenomena for application of nanoparticles in the field of biomedicine. A zeta potential analyzer evaluated the present study surface charge of PoAgNPs, and the obtained graph is shown in Figure 6a. From the graph, the surface charge of synthesized PoAgNPs was found to be −25.7 mV. The DLS measurement of PoAgNPs with an average size of 83.14 nm is shown in Figure 6b. The DLS scale is different because it gives the average size of the particles. That may also be due to the samples' non-homogeneous dispersion of the sample.

Hemolytic Activity of PoAgNPs
It is essential that the nanomaterial intended for medicinal use is mandatorily checked for risk assessment in toxicological aspects to ensure their biocompatibility. Our data showed that the synthesized PoAgNPs possessed a lower lysis profile than the positive control (Figure 7a). Biology 2021, 10, x 11 of 21

Hemolytic Activity of PoAgNPs
It is essential that the nanomaterial intended for medicinal use is mandatorily checked for risk assessment in toxicological aspects to ensure their biocompatibility. Our data showed that the synthesized PoAgNPs possessed a lower lysis profile than the positive control (Figure 7a).

Antibacterial Activity
The antibacterial activity of PoAgNPs is presented in Figure 7b. Based on the results and zone of inhibition, PoAgNPs exhibited outstanding activity against the tested human pathogens. From the data, it is clear that the bactericidal activity progressed according to the dose level of PoAgNPs. The highest zone of inhibition was observed in the following order: M. luteus > M.smegmatis > E. coli > K. pneumoniae > V. cholerae > B. subtilis.

MIC and MBC Analysis
The MIC and MBC values are shown in Table 2. The data inferred that MIC varies by the genera and species. The inhibitory concentration of PoAgNPs, except for B. subtilis

Hemolytic Activity of PoAgNPs
It is essential that the nanomaterial intended for medicinal use is mandatorily checked for risk assessment in toxicological aspects to ensure their biocompatibility. Our data showed that the synthesized PoAgNPs possessed a lower lysis profile than the positive control (Figure 7a).

Antibacterial Activity
The antibacterial activity of PoAgNPs is presented in Figure 7b. Based on the results and zone of inhibition, PoAgNPs exhibited outstanding activity against the tested human pathogens. From the data, it is clear that the bactericidal activity progressed according to the dose level of PoAgNPs. The highest zone of inhibition was observed in the following order: M. luteus > M.smegmatis > E. coli > K. pneumoniae > V. cholerae > B. subtilis.

MIC and MBC Analysis
The MIC and MBC values are shown in Table 2. The data inferred that MIC varies by the genera and species. The inhibitory concentration of PoAgNPs, except for B. subtilis (100 µg/mL), was found to be 25 µg/mL for Gram-positive bacterial pathogens (M. smegmatis and M. luteus), whereas for Gram-negative bacterial pathogens (E. coli, V. cholerae, and K. pneumoniae) it was found to be 50 µg/mL. However, the PoAgNPs significantly inhibited the growth of bacterial pathogens at low concentrations to high concentrations in an impressive manner. Furthermore, the MBC values portrayed the bactericidal effect of PoAgNPs with a value range from 100 to >100 µg/mL.

Confirmation of MIC by Resazurin Dye Assay
Resazurin was used as an indicator in this study. In viable cells, oxidoreductases convert the resazurin salt to resorufin from blue to pink fluorescent. In the 96 well plates, according to the dose level of MIC, the color variation (blue to pink) can be visually observed in Figure 8. This indicated the viable and non-viable cells upon treatment of PoAgNPs.
The MIC and MBC values are shown in Table 2. The data inferred that MIC varies by the genera and species. The inhibitory concentration of PoAgNPs, except for B. subtilis (100 µg/mL), was found to be 25 µg/mL for Gram-positive bacterial pathogens (M. smegmatis and M. luteus), whereas for Gram-negative bacterial pathogens (E. coli, V. cholerae, and K. pneumoniae) it was found to be 50 µg/mL. However, the PoAgNPs significantly inhibited the growth of bacterial pathogens at low concentrations to high concentrations in an impressive manner. Furthermore, the MBC values portrayed the bactericidal effect of PoAgNPs with a value range from 100 to >100 µg/mL. Resazurin was used as an indicator in this study. In viable cells, oxidoreductases convert the resazurin salt to resorufin from blue to pink fluorescent. In the 96 well plates, according to the dose level of MIC, the color variation (blue to pink) can be visually observed in Figure 8. This indicated the viable and non-viable cells upon treatment of PoAgNPs.

Fluorescence-Based Study of PoAgNPs against Bacterial Pathogens
The viability of bacterial cells exposed to PoAgNPs at MIC dosages (E. coli (50 µg/mL), V. cholerae (50 µg/mL), B. subtilis (100 µg/mL), K. pneumoniae (50 µg/mL), M. smegmatis (25 µg/mL), and M. luteus (25 µg/mL)) was observed using fluorescence microscopy. In the analysis, the live cells emitted green and dead cells emitted red fluorescence. This was because live cells uptake AO effectively and emit a green color, whereas dead cells uptake EB and emit red fluorescence (Figure 9).   The in-vitro anticancer potential of the PoAgNPs was evaluated against human adenocarcinoma breast cancer (MDA-MB-231) cell lines by MTT assay. From the analysis, it was inferred that PoAgNPs trigger a pronounced inhibitory activity against the cell line with a gradual decline in cell viability in response to the concentration of PoAgNPs (10-100 µg/mL) with IC 50 91 µg/mL (as depicted in Figure 10).

MTT Assay
The in-vitro anticancer potential of the PoAgNPs was evaluated against human adenocarcinoma breast cancer (MDA-MB-231) cell lines by MTT assay. From the analysis, it was inferred that PoAgNPs trigger a pronounced inhibitory activity against the cell line with a gradual decline in cell viability in response to the concentration of PoAgNPs (10-100 µg/mL) with IC50 91 µg/mL (as depicted in Figure 10).

Dual Staining Assay (AO/EB)
To authenticate that the IC50 concentration of PoAgNPs induced apoptosis, the cells were observed under AO/EB staining. The staining encountered the live and dead cells and differentiated them based on the color (live cells (green); dead cells (red); orange (late apoptotic cells)), as shown in Figure 11.

Dual Staining Assay (AO/EB)
To authenticate that the IC 50 concentration of PoAgNPs induced apoptosis, the cells were observed under AO/EB staining. The staining encountered the live and dead cells and differentiated them based on the color (live cells (green); dead cells (red); orange (late apoptotic cells)), as shown in Figure 11. 3.6. In-Vitro Anticancer Activity of PoAgNPs 3.6.1. MTT Assay The in-vitro anticancer potential of the PoAgNPs was evaluated against human adenocarcinoma breast cancer (MDA-MB-231) cell lines by MTT assay. From the analysis, it was inferred that PoAgNPs trigger a pronounced inhibitory activity against the cell line with a gradual decline in cell viability in response to the concentration of PoAgNPs (10-100 µg/mL) with IC50 91 µg/mL (as depicted in Figure 10).

Dual Staining Assay (AO/EB)
To authenticate that the IC50 concentration of PoAgNPs induced apoptosis, the cells were observed under AO/EB staining. The staining encountered the live and dead cells and differentiated them based on the color (live cells (green); dead cells (red); orange (late apoptotic cells)), as shown in Figure 11. Figure 11. AO/EB dual staining apoptotic assay. Figure 11. AO/EB dual staining apoptotic assay.

Larvicidal Activity of PoAgNPs against II and IV Instars Larvae of C. quinquefasciatus
Under laboratory conditions, we investigated the larvicidal efficacy of PoAgNPs against the II and IV larvae of C. quinquefasciatus. The larvicidal activity was performed with varying concentrations (2, 4, 6, 8, and 10 ppm) of PoAgNPs for 24 h exposure. After the treatment period, the larvae were analyzed for mortality. The obtained mortality data showed that PoAgNPs were toxic against the larvae of C. quinquefasciatus with significant LC 50 values of 1.673 and 2.273 ppm ( Figure 12; Table 3). The larvicidal activity of PoAgNPs depends on the mosquito larvae, genus, and species. Interestingly, in the present study, the minimal concentration of 1.6 ppm of PoAgNPs was required to kill 50% of mosquito larvae within a short period. This shows that PoAgNPs are a potentiated biocide to mitigate the eruption of mosquito larvae (Figure 13).

Larvicidal Activity of PoAgNPs against II and IV Instars Larvae of C. quinquefasciatus
Under laboratory conditions, we investigated the larvicidal efficacy of PoAgNPs against the II and IV larvae of C. quinquefasciatus. The larvicidal activity was performed with varying concentrations (2,4,6,8, and 10 ppm) of PoAgNPs for 24 h exposure. After the treatment period, the larvae were analyzed for mortality. The obtained mortality data showed that PoAgNPs were toxic against the larvae of C. quinquefasciatus with significant LC50 values of 1.673 and 2.273 ppm ( Figure 12; Table 3). The larvicidal activity of PoAgNPs depends on the mosquito larvae, genus, and species. Interestingly, in the present study, the minimal concentration of 1.6 ppm of PoAgNPs was required to kill 50% of mosquito larvae within a short period. This shows that PoAgNPs are a potentiated biocide to mitigate the eruption of mosquito larvae ( Figure 13).

Discussion
In nature, a myriad of plants and microbes cohabit, which is exploited mainly for the welfare of human beings and the environment. In particular, the endophytic fungi produce many bioactive constituents with a potential for pharmacognostic applications [27]. This pipeline explored the nanobiotechnological potential of the endophytic fungi P. oxal- Figure 13. Light microscopic images of control-and PoAgNPs-treated mosquito larvae of C. quinquefasciatus with ×10 magnification.

Discussion
In nature, a myriad of plants and microbes cohabit, which is exploited mainly for the welfare of human beings and the environment. In particular, the endophytic fungi produce many bioactive constituents with a potential for pharmacognostic applications [27]. This pipeline explored the nanobiotechnological potential of the endophytic fungi P. oxalicum strain LA-1 isolated from a medicinal plant. After subsequent isolation and identification, we screened the fractions of solvent extracts. Among the extracts, the highest percentage of compounds was observed in the methanolic extract compared with the other solvent extracts.
Noticeably, major pharmaceutical compounds such as pyrrolo[1, 2-a]pyrazine-1, 4-dione, and hexahydro-3-(2-methyl propyl) were noticed. As reported, these compounds have the potential for significant therapeutic applications [28][29][30]. Moreover, it is a wellestablished fact that the active hydroxyl or amine functional groups in bioactive constituents play a vital role in reducing metal ions. As the reaction proceeds by employing AgNO 3 with the crude extract of P. oxalicum strain LA-1, a dark brown color was observed, indicating the synthesis of PoAgNPs.
Khan et al. [12] demonstrated that in UV-visible spectroscopic analysis of AgNPs, nanoparticles' particle size, morphology, and compositions are directly proportional to the surface plasmon resonance. Herein, the optical properties of PoAgNPs were governed by the factor known as surface plasmon resonance, which generated absorbance spectra at 430 nm [31]. During the reaction, pH plays a crucial role, as a change in the electrical charges present in the culture filtrate substantially increases nanoparticles' growth and yield by altering the reaction kinetics [32]. Our result was consistent with Singh et al. [30]. Qian et al. [33] demonstrated that alkaline pH favored the synthesis of silver nanoparticles when 1 mM silver nitrate was challenged with the cell-free filtrate of E. nigrum. The effect of temperature on the synthesis of silver nanoparticles was also explored in the present study. Even though temperatures at a higher range favored the formation of silver nanoparticles, we annotated that 37 • C was optimal for the stable formation of silver nanoparticles. These features were similar to the reports of Singh et al. [30]. Interestingly, in the FTIR spectrum of PoAgNPs, among the various peaks, a prominent peak located at 1040 cm −1 was found to be associated with the -C-OH of the phenols [34]. This was attributed to the fact that the proteins/alcohol/phenolic groups present in the culture filtrate were mainly responsible for reducing silver ions (Ag+) into nano-sized silver Ag (o) [35][36][37][38]. Additionally, in the XRD spectrum, four major diffraction peaks were located at 27.71 • , 32.20 • , 46.21 • , and 54.78 • , which could be indexed as (111), (200), (220), and (311), which reflected the facecentered cubic crystalline nature of PoAgNPs [39]. As a result, the XRD pattern obtained for the AgNPs showed the crystalline nature of AgNPs with an FCC (Face Centred Cubic) phase that corresponded to several previously reported studies on AgNPs synthesized by fungal extracts (Rafie et al. [37]; Asad et al. [38]). According to an earlier study [40], the peak of the silver ion was produced at 3 KeV, which could help in the reduction of Ag+ to Ag 0 .
The negative charge of synthesized PoAgNPs was mainly due to the culture extract of endophytic fungi containing biomolecules that modify nanoparticle surfaces with their anionic groups [41]. This negative charge bestows high stability and dispersity of NPs without aggregation owing to their strong repulsive forces. Furthermore, the observed hemolytic properties of PoAgNPs could be easily attributed to their size, surface chemistry, and physicochemical properties. The mechanism of PoAgNPs that induces hemolysis is by the interaction of AgNPs with the thiol group of protein and the phospholipids of RBC with a high affinity, which thereby collapses RBCs. Moreover, negatively charged silver ions interact with organic cations in the RBC membrane, which may also contribute to the hemolysis of RBC [42]. Hemolysis is a process of erythrocyte destruction with the release of hemoglobin in the environment [43]. According to the International Organization for Standardization/Technical Report 7406, the acceptable degree of hemolysis for bio-based products was 5% (Ruden et al. [44]). In our study, PoAgNPs demonstrated an adequate hemolysis rate, indicating its biocompatibility and suitability for biomedical applications. Our findings are consistent with the previous research of Sathish Kumar et al. [45].
In the present study, the inhibitory action of PoAgNPs was comparatively low in Gram-positive bacteria than in Gram-negative bacteria. This phenomenon was due to the presence of a thick peptidoglycan layer in which AgNPs could effectively stick to the cell wall of bacteria, thus preventing antibacterial action [46]. The bactericidal effect of AgNPs against pathogenic bacteria was achieved by attaching AgNPs to the bacterial cell surface and membrane, causing intracellular damage to the proteins, DNA, RNA, respiratory chain, and the metabolism, followed by induction of oxidative stress (ROS generation) and, finally, cell death [47]. Hence, we speculated that PoAgNPs may exhibit the above fourth mechanism against the tested pathogens.
The in-vitro cytotoxicity exhibited an effective growth inhibitory activity in human adenocarcinoma breast cancer (MDA-MB-231) cell lines by MTT assay. The PoAgNPs triggered the toxicity by inducing apoptosis in a dose-dependent manner. The mechanistic action of PoAgNPs is executed by induction of ROS, followed by disruption of intracellular organelles, which eventually leads to apoptosis [48]. The color variation is based on the uptake of dyes. Live cells uptake the low molecular weight dye AO, whereas dead cells uptake the high molecular weight dye EB. This phenomenon occurs when cells undergo apoptosis; thus, the cell membrane blebs and leaks, nuclei condense, and DNA fragmentation can be observed [49].
Apart from the antibacterial properties, the biogenic AgNPs have been reported to hold excellent larvicidal properties against disease-transmitting mosquito vectors [50]. Prabakaran and colleagues recorded that silver nanoparticles from Beauveria bassiana against different larval stages of Culex sp. had significant mortality effects, similar to what we found in our current study [51]. Our data clearly showed the possible mechanism of AgNPs against the larvae by breaching the exoskeleton of C. quinquefasciatus mosquito larvae that binds with the phosphorous/sulfur of DNA and protein, which results in the blockade of transcription and translation followed by the internal collapse of cell organelles and, finally, cell death [52].

Conclusions
To summarize, we documented the synthesis of PoAgNPs using a culture filtrate of the endophytic fungi P. oxalicum LA-1 strain. We exclusively characterized the PoAgNPs through UV-vis spectroscopy, FTIR, XRD, and TEM analyses. The synthesized PoAgNPs inhibited human pathogenic bacterial growth, which depicts its potential as an antibacterial agent. PoAgNPs effectively demonstrated the increased inhibitory activity in human adenocarcinoma breast cancer (MDA-MB-231) cell lines by MTT assay with the IC 50 concentration of 91 µg/mL. Moreover, larvicidal screening showed that PoAgNPs are an effective biocide against the disease-transmitting C. quinquefasciatus. Finally, PoAgNPs unveiled significantly less RBC lysis, which denotes its biocompatibility and possibility for medicinal use. Overall, we believe that a sustainable approach was developed to synthesize versatile PoAgNPs with improved physicochemical properties. The synthesized nanomaterials can be developed as an antibacterial/larvicide agent after being subject to in vitro and in vivo pre-clinical studies. Based on the obtained results, we conclude that the synthesized PoAg-NPs could act as a potent antibacterial/antitumor agent after completing the pre-clinical and clinical studies.