*5.3. Other Nanomaterials*

Abdullah et al. introduced the green synthesis method of ZnO@TiO2@SiO<sup>2</sup> and Fe3O4@SiO<sup>2</sup> nanocomposites (NCs) utilizing the novel lichen species *Lecanora muralis* [164]. Lichen specimens were collected from Grdmandil mountain and the chemical compositions of the rock-inhibiting lichen samples were analyzed via XRD assay. The samples comprised quartz, hematite, magnetite, and maghemite Q. Similarly, biomolecules inside the lichen cell extract were determined using gas chromatography-mass spectroscopy, and there were a variety of secondary metabolites present that have many medical applications through their antioxidant, anticancer, analgesic, and antipyretic activities. To synthesize ZnO@TiO2@SiO2, 2 g of *L. muralis* (LM) was mixed with 30 mL distilled water and the mixture was boiled at 80 ◦C for 1 h then filtrated. Next, 20 mL LM filtrate was mixed with 0.5 g ZnCl2, 1.5 g TiO(OH)<sup>2</sup> (titanyl hydroxide), and 2.5 g Na2SiO<sup>3</sup> at pH 8 and 80 ◦C for 5 h under stirring conditions. Similarly, Fe3O4@SiO<sup>2</sup> NCs were formed by mixing 20 mL LM filtrate and 2 g Na2SiO<sup>3</sup> with 0.7 g FeCl<sup>2</sup> and 1.2 g FeCl<sup>3</sup> at pH 9 and 80 ◦C for 5 h under stirring conditions. After the incubation period, NP precipitates were filtrated, washed with hot distilled water to discard any impurities, and then dried. Physical and chemical analyses of the ZnO@TiO2@SiO<sup>2</sup> and Fe3O4@SiO<sup>2</sup> NCs showed that *L. muralis* has the potential to fabricate NCs from their bulk materials. XRD demonstrated that the biosynthesis of ZnO@TiO2@SiO<sup>2</sup> and Fe3O4@SiO<sup>2</sup> NCs generates crystallinity nanoforms of 55 and 53 nm, respectively. Furthermore, the authors reported that Fe3O<sup>4</sup> NPs coated the surface of the silica oxide nanoparticles. SEM micrographs of ZnO@TiO2@SiO<sup>2</sup> and Fe3O4@SiO<sup>2</sup> NCs revealed that these NCs were spherical and had a nanosize range of 55–90 and 50–85 nm, respectively. Some agglomeration was observed by SEM in both types of NC. EDX and elemental mapping showed that ZnO@TiO2@SiO<sup>2</sup> NC was synthesized from Zn, O, Ti, and Si with no further elements, indicating the purity of the formed nanostructure; however, the compositional elements of Fe3O4@SiO<sup>2</sup> NCs involving Fe, O, and Si indicated the binding of Fe3O<sup>4</sup> NPs on the surface of SiO<sup>2</sup> NPs (Table 1).

Bimetallic NPs (Au–Ag NPs) were extracellularly synthesized using *Cetraria islandica* [144]. In brief, 1 mL lichen extract was mixed with 10 mL of 1.5 mm HAuCl<sup>4</sup> and AgNO<sup>3</sup> solutions and 0.5 m NaOH solution (pH 10) and incubated for 30 min at 80 ◦C under continuous mixing and stirring conditions. The reaction was repeated with different molar ratios of the Ag and Au solutions (1:1, 1:2, and 2:1), and the resulting bimetallic NPs are defined as Ag50Au50, Ag33Au67, and Ag67Au33, respectively. Only one absorption band appeared between the SPR of both monometallic Au- and Ag-NPs, and as Au content increased, the absorbance band redshifted. UV absorbance peaks of Ag50Au50, Ag33Au67, and Ag67Au33 were 412, 519, and 523 nm, respectively. This finding implied that bimetallic NPs may have only one alloy. FTIR spectra peaks for Ag33Au67 were at 1383 cm−<sup>1</sup> , which relate to C=O of carboxylic acid and methyl interactions in large, branched molecules that play an important role in stabilizing and capping NPs. TEM images of Ag33Au67 bimetallic NPs showed that these NPs were spherical and polygonal with nanosizes of 6 and 21 nm, respectively. The narrow particle size indicates a large active surface area for catalytic activity.

Esmaeili and Rajaee explored an eco-friendly synthesis method using lichen usnic acid as a nanoparticle mediator to produce nanohyaluronic acid [146]. In brief, usnic acid (UAL) was extracted from *Aspicilia lichens* with acetone using a Soxhlet apparatus for 8 h. The FTIR spectrum of purified UAL was similar to the standard usnic acid spectrum. The stretching of O–H in the Ar–OH intramolecular hydrogen bond showed strong bands at 3421 and 3371 cm−<sup>1</sup> . Additionally, (–CH3) of the alkane groups in UAL had peaks at 2925 and 2854 cm−<sup>1</sup> caused by stretching of the C–H bond. The presence of the C=C group in UAL was indicated by a peak at 1658 cm−<sup>1</sup> , which is due to the presence of the aryl group. The aromatic methyl ketone at 1625 cm−<sup>1</sup> is related to the hydrogen bonds. Similarly, the conjugate cyclic ketone group is confirmed by a peak at 1739 cm−<sup>1</sup> . UAL also showed hydroxyl phenolic signals at 3095 cm−<sup>1</sup> , which is likely due to the stretching of a symmetrical or nonsymmetrical group C–O–C that bonded to an aryl-alkyl-ester at

1265 and 1074 cm−<sup>1</sup> . SEM micrographs demonstrated that UAL NPs were spherical with a mean size of 29–89 nm and no agglomeration. For the preparation of nanohyaluronic acid, 2 g hyaluronic acid (produced by mixing *Bifidobacterium* sp. and the solution of UAL extract) was added to 50 mL distilled water and 200 mL of a UAL solution with acetone and methanol (5:2) at 50 ◦C for 48 h with stirring. The NPs were then collected by centrifugation at 12,000 rpm for 30 min. SEM micrographs showed that hyaluronic NPs have an average nanosize of 55 nm. FTIR spectra of nanohyaluronic acid showed that the usnic acid extracted from *Aspicilia* sp. has strong redox activity that enables these compounds to reduce the hyaluronic acid into their nanoform (Figure 4).

**Figure 4.** Types of nanoparticles (NPs) synthesized by lichen species.

#### **6. Prospective Applications of Lichen-Based Nanoparticles**

### *6.1. Antimicrobial Activity*

Khandel et al. studied the inhibitory activity of Ag-NPs synthesized by *Parmotrema tinctorum* and the activity of silver nitrate and lichen extract against five pathogenic bacteria—*Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Escherichia coli*, *Bacillus subtilis*, and *Klebsiella pneumoniae*—for 24 h at 35 ◦C using the agar well diffusion method [149]. Ag-NP (10, 30, and 50 µL) was the most potent antibacterial agent, causing greater inhibition of bacterial growth compared with silver nitrate and lichen extract. Ag-NPs suppressed the growth of both Gram-negative and Gram-positive bacteria at the three concentrations tested. At the highest concentration of Ag-NPs (50 µL), the greatest inhibition zone (IZ) was detected against *P. aeruginosa* (17 ± 0.50 mm), *K. pneumoniae* (14 ± 0.10 mm), and *E. coli* (11 ± 0.10 mm), while the lowest IZ was observed against *B. subtilis*(8 ± 0.30 mm) and *S. aureus* (7 ± 0.30 mm). The authors reported that Ag-NPs were more effective at inhibiting Gram-negative bacteria than Gram-positive bacteria due to the difference in the cell-wall structure of the bacteria; Gram-positive bacteria have a thicker cell wall than Gram-negative bacteria, and hence, penetration of their cell wall is difficult. Furthermore, the authors conclude that the mode of action of Ag-NPs against bacteria is via their ability to change the membrane structure and permeability, leading to bacterial death.

Iron oxide-NPs (0.075–0.00046875 mg/mL) bioformed by *Ramalina sinensis* significantly inhibited the bacterial growth of both *P. aeruginosa* and *S. aureus* after incubation for 24 h at 37 ◦C [165]. Iron oxide-NPs at 0.075 mg/mL exhibited the highest antibacterial activity, while 0.0075 and 0.000234375 mg/mL of iron oxide-NPs were the lowest inhibitory concentrations of NPs against both *P. aeruginosa* and *S. aureus*, respectively. The antibacterial activity of iron oxide-NPs was almost equivalent to that of tetracycline. The authors suggested that the expected killing mechanism of NPs against bacteria may be related

to the electrostatic activity between iron oxide-NPs and the bacterial membrane. This interaction might result in the release of the iron ions by the NPs, and these ions can then interact with the thiol group on membrane proteins, causing bacterial membrane oxidation, subsequent stimulation of reactive oxygen species (ROS), loss of membrane permeability, disruption of cell membrane respiration, and ultimately, bacterial death.

Abdullah et al. studied the antibacterial and antifungal activities of both ZnO@TiO2@SiO<sup>2</sup> and Fe3O4@SiO<sup>2</sup> NCs synthesized by *Lecanora muralis* against *S. aureus*, *E. coli*, *Pseudomonas* sp., and five species of fungi, i.e., *Candida albicans*, *Candida* spp., *Aspergillus flavus*, *Aspergillus niger*, and *Aspergillus terrus*, utilizing both disk diffusion and agar well diffusion assays and compared the results with those of lichen extract alone [164]. Both NCs showed higher inhibitory activity than lichen extract alone against bacterial and fungal species, with the exception of the three species of the genus *Aspergillus* (zero inhibition zone). Fe3O4@SiO<sup>2</sup> exhibited the highest bioactivity among the treatments, suggesting more bioactive molecules were precipitated on Fe3O4@SiO<sup>2</sup> NCs than on ZnO@TiO2@SiO<sup>2</sup> NCs. The authors noted that the increased antioxidant molecules adsorbed on Fe3O4@SiO<sup>2</sup> NCs contributed to the long-term stabilization of NCs against decomposition and deformation conditions.

Lichen ethanolic extract (*Parmotrema clavuliferum*) and the corresponding lichensynthesized Ag-NPs were investigated as an antibacterial treatment in a recent study by Alqahtani et al. [158]. Biogenic Ag-NPs showed a significant inhibitory effect against *P. aeruginosa* (11.5 ± 0.9 mm), *Streptococcus faecalis* (7.6 ± 1.7 mm), *B. subtilis* (8.1 ± 1.5 mm), and *S. aureus* (8.1 ± 1.5 mm). However, the ethanolic extract of the lichen caused had the highest zone of inhibition (19.8 ± 0.9 mm) against *B. subtilis* and the lowest zone of inhibition (3.6 ± 0.9 mm) against *S. aureus*. Furthermore, *S. faecalis* and *P. aeruginosa* showed inhibition zones of 15.5 ± 1.6 and 13.8 ± 0.9 mm, respectively, after spiking with lichen extract. The authors suggested that Ag-NPs could have this bactericidal effect due to one or more of the following actions: Ag-NPs may induce cell lysis, hinder transduction, change membrane permeability, or destroy the bacterial genome through DNA fragmentation.

Ag-NPs produced using four lichens, *Parmeliopsis ambigua*, *Punctelia subrudecta*, *Evernia mesomorpha*, and *Xanthoparmelia plitti*, were screened for antibacterial activity against several Gram-negative and Gram-positive bacteria, including *Pseudomonas aeruginosa*, *Escherichia coli, Proteus vulgaricus, Staphylococcus aureus*, *Streptococcus pneumoniae*, and *Bacillus subtilis* [153]. The disk diffusion method was used for screening with 0.02 mg of the produced Ag-NPs. *Pseudomonas putida* was the most susceptible to Ag-NPs synthesized by *X. plitti* (2.3 cm), followed by *Pseudomonas aeruginosa* with Ag-NPs synthesized by *E. mesomorpha* extract (1.9 cm) and *Bacillus subtilis*, which was the least susceptible with Ag-NPs synthesized by *X. plitti* extract (1.3 cm).

The antibacterial activity of Ag-NPs synthesized using an aqueous extract of *Ramalina dumeticola* were examined against four Gram-positive pathogenic bacteria (*Staphylococcus epidermidis*, methicillin-resistant *Staphylococcus aureus* (MRSA), *Bacillus subtilis*, and *Streptococcus faecalis*) and four Gram-negative strains (*Proteus vulgaris*, *Pseudomonas aeruginosa*, *Serratia marcescens*, and *Salmonella typhi*) by applying the disk diffusion method [154]. Gentamycin (30 µg disks) was used as a positive control, and the negative control was sterile distilled water. A total of 10 microliters of 100 µg/mL of the Ag-NPs solution was applied to the sterile disks on agar plates, and inhibition zones (IZs) were measured after incubation for 18–24 h at 37 ◦C. The results demonstrated the potential of the Ag-NPs as a bactericidal factor. The Ag-NPs were effective against both types of bacteria but showed more efficacy towards Gram-negative bacteria than Gram-positive bacteria. The largest IZ was observed in *Proteus vulgaris* (10.5 ± 0.7 mm), followed by *Pseudomonas aeruginosa* (9.5 ± 3.5 mm) and MRSA (9.5 ± 0.7 mm). The Ag-NPs were less effective in *Salmonella typhi* (IZ diameter of 8.5 ± 2.1 mm) and *Serratia marcescens* (7.5 ± 0.7 mm). *Bacillus subtilis* and *Streptococcus faecalis* had identical IZ (7.5 ± 0.0 mm), while the least inhibitory effect of the Ag-NPs was observed on *Staphylococcus epidermidis* with an IZ of 7 ± 0.0 mm. The same concentration of aqueous extract (100 µg/mL) resulted in a lower inhibition zone diameter of 6 mm against all tested microbes compared with the IZ diameters produced

with Ag-NPs. This suggested that the Ag-NPs have higher antibacterial activity than the lichen extract. The authors hypothesize that the increased susceptibility of Gram-negative bacteria to Ag-NPs compared with Gram-positive bacteria is likely due to the thinner peptidoglycan layer of Gram-negative bacteria, which provides the Ag-NPs with better anchoring and penetration of the cell wall.

Ag-NPs synthesized by Usnea longissima were evaluated for antimicrobial potency against six-Gram positive bacteria (*Staphylococcus aureus*, *Streptococcus mutans*, *Streptococcus pyogenes*, *Streptococcus viridans*, *Corynebacterium diphtheriae*, and *Corynebacterium xerosis*) and three Gram-negative bacteria (*Escherichia coli*, *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa*) by agar well diffusion method [136]. The bacteria were incubated with Ag-NPs for 24 h at 37 ◦C. A negative control (DMSO) and positive controls (ciprofloxacin (5 µg/disk) for Gram-positive bacteria and Gentamicin (10 µg/disk) for Gram-negative strains) were used to compare the inhibitory activity of NPs. The Ag-NPs displayed the highest antibacterial efficiency against *E. coli* and *K. pneumoniae* with IZ diameters of 20.8 ± 0.02 and 16 ± 0.31 mm, respectively. In contrast, *S. mutans* (6.5 ± 0.89 mm), *C. diphtheriae* (6.2 ± 0.37 mm), and *P. aeruginosa* (7 ± 0.31 mm) were not affected by the Ag-NPs. The Ag-NPs were suggested to have a low antibacterial effect on the basis that the antibacterial effect can be amplified by reducing the NP size and hence increasing the surface area. As the surface area of the Ag-NPs increases, contact with microorganisms improves, which mediates penetration of the particles into the bacterial cell membrane or attachment to the bacterial surface. When silver ions reach the bacterial cytoplasm, they can denature the ribosome, thus directing the suppression of cell enzymes and proteins. Consequently, the metabolic function of the bacterial cell will be disrupted and the cell will undergo apoptosis. The authors reported that the lethal effect of Ag-NPs against bacteria can be achieved by different mechanisms including (i) interfering with cell wall, (ii) suppression of protein synthesis, and (iii) disruption of transcription and primary metabolic processes.

Kumar et al. studied the synergistic antibacterial effect of the extracts of two lichens, *Parmotrema pseudotinctorum* and *Ramalina hossei*, combined with chemically synthesized Ag-NPs, against several strains of Gram-positive and Gram-negative bacteria known to cause food poisoning [171]. The tested strains *Staphylococcus aureus*, *Bacillus cereus*, *Escherichia coli*, and *Salmonella typhi* were treated with the lichen extracts and Ag-NPs individually and with a combination of both, utilizing the agar well diffusion method. On Muller-Hinton agar plates, bacterial broth cultures (10<sup>8</sup> cells/mL) were swabbed then wells of 6-mm diameter were loaded as follows: lichen extracts (10 mg/mL in DMSO), Ag-NPs (1 mg/mL in DMSO), standard (chloramphenicol, 1 mg/mL), a combination of lichen extract and Ag-NPs (1:1 ratio), and control (DMSO). The plates (two replicates of each) were incubated at 37 ◦C for 24 h and the IZs were measured and the mean value was calculated for each sample. According to the IZs, the lichen extracts were more effective than the Ag-NPs alone on most plates. The Ag-NPs were more effective against Gram-negative bacteria than Gram-positive bacteria. However, the combination of the lichen extracts and Ag-NPs showed more bacterial inhibition than that of the extract alone or the NPs alone. After exposure to the combined treatment, *S. typhi* had an IZ of 2.8 cm, followed by *E. coli* (2.6 cm), *B*. *cereus* (2.1 cm), and *S. aureus* (1.9 cm). The enhanced antibacterial activity of the combined treatment might be attributed to the presence of effective secondary metabolites in the lichen extracts, and also the smaller-size-to-large-surface-area ratio of Ag-NPs. In the lichen extracts experiments, Gram-positive bacteria were more affected than Gram-negative bacteria. However, in the combined treatment assays, the antibacterial activity was more pronounced against Gram-negative bacteria. The authors attributed this to Gram-negative bacteria being naturally more resistant due to their thick outer membrane that prevents harmful substances from entering the cell. This barrier comprises an exterior lipopolysaccharide layer and a thin layer of peptidoglycan at the interior. TEM imaging confirmed that Ag-NPs can be effective bactericidal agents by rupturing the bacterial membrane even at low concentrations.

#### *6.2. Antioxidants*

The antioxidant activity of biomatrix loaded with Au-NPs synthesized by *Acroscyphus sphaerophoroides* and *Sticta nylanderiana* was screened by Debnath et al. using a modified diphenylpicrylhydrazyl (DPPH) method. Powdered samples of 2 and 5 mg were treated in two separate test tubes with 3 mL of 100 M methanolic solution of DPPH. The surface reaction for both mixtures was amplified by sonicating them in the dark. To confirm time-dependent DPPH scavenging, centrifugation was performed and the absorbance of the supernatants over time was measured at 517 nm with DPPH as a reference and a gap of 15, 30, 45, and 60 min. Measurement of the scavenging potential (SC50) of biomatrix-loaded Au-NPs synthesized by *A. sphaerophoroides* and *S. nylanderiana* is achieved via a similar process, where absorbances are documented at 30 min after administering 1, 1.5, 2, 2.5, 3 mg and 1, 3, 5, 7, 10 mg of the samples, respectively. The concentrations of gold-NPs synthesized by *A. sphaerophoroides* and *S. nylanderiana* responsible for scavenging of 50% of DPPH (SC50) were 1.66 and 4.48 mg, respectively, suggesting biogenic gold-NPs were potent antioxidant agent [159].

An extract of the lichen *Parmelia sulcata* was exploited for the biological formulation of Au-NPs [160], and the resulting Au-NPs and *P. sulcata* extract were tested for their free radical scavenging potential in antioxidant bioassays involving DPPH and hydrogen peroxide. For the DPPH method, 2.96 mL of 0.1 mM solution of DPPH was added to 0.4 mL of the extract or Au-NPs at different concentrations (250, 500, 750, and 1000 µg/mL) and incubated under dark conditions at ambient temperature for 30 min. The absorbance was recorded at 517 nm and used to calculate the percentage inhibition of scavenging potential. For the hydrogen peroxide scavenging test, 40 mM H2O<sup>2</sup> solution was prepared in phosphate buffer at pH 7.4 and then several concentrations (250, 500, 750, 1000 µg/mL) of extracts and Au-NPs were added and incubated for 10 min at room temperature. The absorbance was measured at 230 nm and was subsequently used to determine the percentage of inhibition. The outcomes of these bioassays consolidated the ability of the lichen extract and Au-NPs to scavenge free radicals; the IC<sup>50</sup> values of DPPH were 1020 and 815 µg/mL and the IC<sup>50</sup> values of H2O<sup>2</sup> were 694 and 510 µg/mL, respectively. These results indicated that the Au-NPs had greater potential for free radical scavenging (FRS) compared with the lichen extract. In addition, the FRS activity of both lichen extract and Au-NPs appears to be concentration dependent.

#### *6.3. Other Applications*

A recent study used ZnO-NPs biosynthesized by *Ramalina fraxinea* extract as a cytotoxic agent for human neuroblastoma cells [169]. The study focused on evaluating the neurotoxicity and neuroprotective effect of lichen-synthesized ZnO-NPs against SHSY-5Y human neuroblastoma cells. Several concentrations of ZnO-NPs were prepared to identify the cytotoxic doses of these NPs. A concentration of 25 µg/mL ZnO-NPs significantly increased the cell viability (*p* < 0.05) when compared with the control group. However, a lower concentration (5 µg/mL) of ZnO-NPs did not affect SHSY-5Y cells. ZnO-NPs at 50, 100, 200, and 400 µg/mL caused a marked reduction (*p* < 0.001) in cell viability, compared with those of the control group. To estimate the neuroprotective effect of ZnO-NPs, the authors exploited the ability of hydrogen peroxide to induce apoptosis of SHSY-5Y cells via oxidative stress; for this purpose, 300 µM H2O<sup>2</sup> was used to treat the cells. This treatment resulted in a significant reduction (*p* < 0.001) of cell viability compared with the control group. ZnO-NPs (at all tested doses) did not increase H2O2-induced death of the SHSY-5Y cells. Moreover, the higher doses (100, 200, and 400 µg/mL) of ZnO-NPs markedly reduced (*p* < 0.001) the cell viability compared with the H2O<sup>2</sup> group. In summary, ZnO-NPs at high doses (≥50 µg/mL) can induce neurotoxicity in SHSY-5Y neuroblastoma cells but provide neuroprotection against the neurotoxic effect of hydrogen peroxide at a low to moderate doses (25 µg/mL).

Iron oxide-NPs fabricated by *Ramalina sinensis* were able to remove lead and cadmium (82 and 77%, respectively) from aqueous solution at an initial concentration of 50 mg/L and with pH in the range of 5–4, indicating the potential of these NPs to be heavy metal eliminators [163].

Çıplak et al. conducted the first study on the catalytic activity of biogenic monometallic NPs (Ag- and Au-NPs) and bimetallic NPs (Au-Ag NPs) synthesized by *Cetraria islandica* [144]. Bimetallic NPs showed higher catalytic activity than monometallic NPs for the reduction of nitrophenols (4-nitrophenol; 4-NP) to aminophenols (4-aminophenol; 4-AP) with sodium borohydride (NaBH4). The higher catalytic performance of Au-Ag NPs might be attributed to the higher ionization potential of Au (9.22 eV) than Ag (7.58 eV), which causes electronic charge transfer from Ag to Au and results in an increase in the electron density on the NP surface. Similarly, Au-NPs exhibited better catalytic potentiality than the Ag-NPs.

The reducing power, hydrogen peroxide scavenging ability, and antidiabetic activities of Ag-NPs synthesized by both *Parmelia perlata* aqueous extract and their purified glycoside and alkaloid fractions were screened by Leela and Anchana [150]. Biogenic Ag-NPs generated from lichen fraction biomolecules have significant antidiabetic potential, reducing power, and free radical scavenging ability, compared with the Ag-NPs fabricated by lichen aqueous extract. The antidiabetic properties of the biogenic Ag-NPs were tested using an alpha-amylase inhibition assay and the percentage inhibition of alpha-amylase was 11.11% for Ag-NPs synthesized by lichen aqueous extract, and 51.85 and 29.62% for the Ag-NPs fabricated by the glycoside fraction and alkaloid fraction, respectively. This indicated that glycoside-mediated-Ag-NPs exhibited the strongest antidiabetic activity. The authors suggest that these biogenic Ag-NPs may lead to improvements in type 2 diabetic disease. Furthermore, the reducing activity of the same Ag-NPs was explored in a reducing power assay in which Ag-NPs interact with potassium ferricyanide (Fe3+), leading to the generation of potassium ferricyanide (Fe2+), which then reacts with ferric chloride to form a ferric-ferrous complex that is readily detected by UV spectrophotometer. Glycoside-mediated-Ag-NP had the greatest reducing activity among the three types of Ag-NPs (absorbance of 0.771, compared with 0.639 and 0.4 for Ag-NPs fabricated utilizing lichen aqueous extract and the alkaloid fraction, respectively). The hydrogen peroxide scavenging ability of the Ag-NPs was also examined. Glycoside-mediated-Ag-NP had the highest scavenging activity (28.89%), compared with Ag-NPs biofabricated by alkaloid fraction (21.86%) and lichen aqueous extract (7.21%).

*Parmelia sulcata* extract (PSE) and PSE-synthesized Au-NPs were investigated for their mosquitocidal activity against *Anopheles stephensi* and *Anopheles aegypti* mosquito larvae, pupae, adults, and egg hatching [160]. Varying concentrations of the lichen extract (75, 150, 225, 300, and 375 ppm) were tested and deemed toxic against larval instars I–IV and pupae of *A. stephensi* and *Anopheles aegypti*. The registered lethal concentration<sup>50</sup> (LC50) values of instars of *A. stephensi* were: 172.16 ppm (I), 201.39 ppm (II), 219.04 ppm (III), 243.89 ppm (IV), and 288.03 ppm (pupae), and the ones for *Anopheles aegypti* were 281.71 ppm (I), 244.46 ppm (II), 283.90 ppm (III), 330.35 ppm (IV), and 346.99 ppm (pupae). The green-synthesized Au-NPs showed exceptionally high activity against larvae and pupae. At concentrations of 15, 30, 45, 60, and 75 ppm, the Au-NPs presented LC<sup>50</sup> values of 29.82 ppm (I), 33.83 ppm (II), 37.55 ppm (III), 44.26 ppm (IV), and 50.44 ppm (pupae) for *A. stephensi*, and 34.49 ppm (I), 38.72 ppm (II), 44.72 ppm (III), 51.41 ppm (IV), and 59.00 ppm (pupae) for *A. aegypti*. For the adulticidal experiments, the PSE concentrations were 25, 50, 75, 100, and 125 ppm, while those of the Au-NPs were 10, 20, 30, 40, and 50 ppm. The LC<sup>50</sup> and LC<sup>90</sup> values of PSE and Au-NPs for *A. stephensi* were 59.35 and 132.80 ppm, and 22.43 and 49.02 ppm, respectively. For *A. aegypti*, the LC<sup>50</sup> and LC<sup>90</sup> values of PSE and Au-NPs were 70.16 and 149.66 ppm, and 24.55 and 52.74 ppm, respectively. Multiple concentrations of PSE and Au-NPs (60, 120, 180, 240, 300, 360, and 420 ppm) were tested for their ovicidal effects, and it was concluded that both *A. stephensi* and *A. aegypti* hinder complete egg hatchability at 360 and 240 ppm, respectively. The Au-NPs impose a high toxicity risk on *A. stephensi* and *A. aegypti*. Importantly, the synthesized Au-NPs worked at a far higher efficacy when compared with the PSE. These experiments proved that PSE-synthesized

Au-NPs have a markedly successful mosquitocidal effect against *Anopheles.* The study was concluded to provide evidential support for the use of PSE and Au-NPs as a solution towards mosquito-manifested environments (Figure 5).

**Figure 5.** Application of lichen-based nanoparticles.

#### **7. Analysis and Characterization of Nanoparticles**

Physicochemical characterization analyses of NP samples are the initial and most significant step following the fabrication process of NPs. These analyses are required to confirm the synthesis of NPs and their unique properties such as increased surface area, stability, crystallinity, charge, dispersion, magnetic, thermal, and optical properties, and morphological features such as shape and size. The techniques utilized include spectroscopic analyses such as UV–visible spectroscopy, FTIR, zeta potential, dynamic light scattering, and nuclear magnetic resonance spectroscopy. These spectroscopic methods estimate the corresponding wavelength ranges of NPs, the functional groups surrounding NPs, and evaluate the charge and hydrodynamic diameter of NPs. X-ray-based analyses such as XRD, X-ray photoelectron spectroscopy (XPS), and energy-dispersive spectroscopy (EDAX or EDS) are performed to reveal the chemical composition and crystal structure and

phase of the NPs. Microscopic analyses such as TEM, SEM, high-resolution TEM (HRTEM), and atomic force microscope (AFM) are used to demonstrate the morphological features of NPs [153].
