4.1.5. Lipids

Mannosylerythritol lipids were used as a reducing and stabilizing agent in the green synthesis of Ag-NPs [100]. The process commenced with the addition of 0.01 g mannosylerythritol lipids to 1 mL acetone diluted with 10 mL dechlorinated water; pH of the whole solution was adjusted to 7 utilizing 0.1 M sodium hydroxide. The solution was added dropwise to 100 mL of 2 mM silver nitrate solution and kept at room temperature with continuous stirring. The mixture changed from pale-yellow to brownish-red, and the UV–Vis absorption spectrum of the synthesized Ag-NPs was recorded at 430 nm. This confirmed that mannosylerythritol lipids were effective as reducer and stabilizer agents in the formulation of Ag-NPs. An energy dispersive spectroscopy (EDS) instrument equipped with the SEM was used to determine the chemical composition, size, and morphology of Ag-NPs. The structure of the Ag-NPs was perceived by TEM after dispersing powdered NPs in methanol and sonicating the solution. The TEM structure provided more information about the crystallinity and average size of the Ag-NPs.

X-ray diffraction (XRD) of the produced Ag-NPs showed four characteristic peaks of 28.4◦ , 33.2◦ , 47.4◦ , and 56.3◦ at 2θ, which correspond to the lattice planes (111), (200), (220), and (311), respectively, confirming the crystalline and face-centered cubic (fcc) structure of the NPs. Meanwhile, the FTIR spectrum of the mannosylerythritol lipids Ag-NPs demonstrated significant peaks at 3337, 2923, 1742, 1562, 1344, 1093, 718, and 534 cm−<sup>1</sup> , which indicate the presence of various functional groups in the mannosylerythritol glycolipid capping the Ag-NPs. The peak at 3337 cm−<sup>1</sup> may be due to –OH from polysaccharides, while the peak at 2923 cm−<sup>1</sup> might indicate (C–H) stretching of alkanes. The strong band at 1562 cm−<sup>1</sup> could be due to the carbonyl stretching vibration. The peaks at 1466 and 1344 cm−<sup>1</sup> can be assigned to (C–N) and (C–C) stretching vibration of aromatic and aliphatic amines, while the band at 1093 cm−<sup>1</sup> could be assigned to (C–O) of alkoxy groups, and peaks at 718 and 534 cm−<sup>1</sup> to CH<sup>2</sup> groups.

A different study used *Lactobacillus casei* to synthesize of Au-NPs and the *L. casei* components were compared before and after the addition of auric acid (0.5 mM K[AuCl4]) [101]. The levels of unsaturated lipids decreased significantly after the addition of auric acid. Moreover, the formation of Au-NPs caused a reduction in the levels of diglycosyldiacylglycerol (DGDG) and triglycosyldiacylglycerol (TGDG). DGDG extracted from *L. casei* induced the formation of Au-NPs, suggesting that these glycolipids can act as potent reducing agents for the conversion of Au(III) to Au(0) and that results in the formation of small NPs.

#### 4.1.6. Vitamins

The utilization of vitamin B2 as a reducing and capping agent in the green synthesis of Ag and Pd nanowires and nanorods is a distinctive technique in the field of green nanotechnology [102]. Ascorbic acid (vitamin C) is used as a reducing factor in combination with chitosan as a stabilizing agent to fabricate sodium alginate-silver NPs [103]. Malassis et al. demonstrated a prompt and effective method to fabricate Au-NPs and Ag-NPs by exploiting ascorbic acid as a reducing and stabilizing agent [104]. The size of the NPs produced was 8–80 nm for Au-NPs and 20–175 nm for Ag-NPs. The method yielded versatile NP surface modification with a large variety of water-soluble surfactants that can be neutral, positively, or negatively charged. Ahmed et al. reported that ascorbic acid in *Desmodium triflorum* was the predominant biomolecule in the reduction process for Ag-NPs [105].

Production of Se-NPs coated with ascorbic acid was achieved through the bioreduction of selenite (Na2SeO3) [106]. Selenite was mixed with ascorbic acid and the mixture turned orange red after 30 min, confirming the fabrication of Se-NPs. The produced Se-NPs were analyzed by TEM and dynamic light scattering (DLS) and were observed to have an average size of 23 ± 5.0 nm. These NPs were shown to be an excellent candidate for radiopharmaceutical imaging techniques used in the diagnosis of liver and kidney cancers.

Another important vitamin exploited for the synthesis of NPs is vitamin B12. To synthesize Ag-NPs, Au-NPs, and Pd-NPs, vitamin B12 solution was mixed with silver nitrate, gold (III) chloride, and palladium acetate solutions, respectively [107]. All mixtures were tested in the presence and absence of microwave (MV) irradiation. The results exhibited that in the absence of MV irradiation, vitamin B12 did not reduce bulk material to their nanoform. However, MV irradiation enhanced the reduction ability of vitamin B12 to fabricate metals into NPs. XRD analysis of the resultant metallic NPs confirmed the efficiency of this vitamin as a reducing agent. The morphological features of the synthesized Ag-NPs, Au-NPs, and Pd-NPs were examined by using SEM and TEM techniques, and large aggregates with irregular shapes and diameters in the range 70–600 nm were observed. Ag samples treated with MW irradiation for 6 min produced NPs with diameters less than 30 nm. While Au samples treated with MV irradiation for 3 min showed irregular shapes and small-size particles with an average diameter of 40 ± 11.7; larger Au NPs with a diameter > 500 nm were observed after a longer period of irradiation (i.e., 6 min). Pd samples irradiated with MV for 3 min resulted in NPs with an average size of 40.2 ± 7.3 nm, whereas that irradiated with MV for 6 min produced two different diameters of 43.9 ± 7.1 and 6.6 ± 2.1 nm. The NPs were spherical, triangular, and decahedron shaped. It was concluded that MV irradiation duration is the key to mitigate noble NPs size.

#### 4.1.7. Secondary Metabolites

Secondary metabolites of different microorganisms, plants, and animal collagen waste were noted to have several properties that enhance the synthesis of NPs and could potentially be deployed in major pharmaceutical studies. Some of the notable secondary metabolites that serve as NP stabilizers include alkaloids, cardiac glycosides, flavonoids, phenols, tannins, and terpenoids [108–110]. Of these compounds, flavonoids are the most utilized secondary metabolites for green synthesis due to their practical structure and the favorable qualities they provide for human health. Pertaining to the flavonoid family are anthocyanins, which have been thoroughly investigated for their antioxidant activity [110]. One study tested the effects of anthocyanins as secondary metabolites on the green synthesis of Ag-NPs by using an aqueous extract of saffron wastage and reported a marked reduction of silver ions and antibacterial activity against several bacterial strains [109].

A study on Ag-NP synthesized using an aqueous extract of *Pteris tripartita* proved the anti-inflammatory activity of flavonoids-coated Ag-NPs by conducting an in vivo investigation on mice with edema, and reported a success rate of nearly 60% [111]. These findings provide an optimistic outlook for the future of NPs in biotechnology and drug discovery applications since they present an efficient way of producing metal NPs without chemical stabilizers or reducers through the use of abundant and natural compounds such as flavonoids, phenols, tannins, terpenoids, reducing sugars, and proteins.

## *4.2. Living Organisms-Mediated Fabrication of NPs*

Many micro- and macro-organisms are used as biofactories to produce NPs with unique physicochemical and biological activities.

#### 4.2.1. Plants

Plant-mediated fabrication of NPs, or phytonanotechnology, is a recognized branch of green synthesis of NPs due to being an eco-friendly, low-cost, rapid, and simple method. Other beneficial features of phytonanotechnology processes are their scalability, bioactivity, biocompatibility, and broad medical applicability [112]. Plant extracts act as reducing and capping agents for the synthesis of many types of NPs [33]. Different parts of plants, including leaves, fruits, stems, seeds, and roots, showed their reducing ability during the synthesis of metallic NPs [113,114]. Singh et al. successfully synthesized Au-NPs and Ag-NPs using *Panax ginseng* leaf and root extracts within 3 and 45 min at 80 ◦C [115]. Saratale et al. fabricated silver nitrate into Ag-NPs using *Acacia nilotica* leave extract as reducing and stabilizing agents to investigate their antineoplastic, free radical scavenging activity and sensing potency for H2O<sup>2</sup> [116]. The scholars reported that Ag-NPs formed within 20 min of mixing 10 mL of plant leave extract to 100 mL of 1 mM AgNO<sup>3</sup> solution. The resultant NPs have a spherical shape and nanosize range of 5 to 30 nm.

Krishnan et al. biosynthesized Ag-NPs from *Piper nigrum* extract and investigated their antitumor activity [117]. TEM images revealed that the Ag-NPs were spherical with a size of 20 nm. The cytotoxicity of Ag-NPs and *Piper nigrum* extract at various concentrations in the range of 10–100 µg/mL was investigated against breast and liver cancer cell lines (MCF-7 and HepG2 cells, respectively) and confirmed their potent cytotoxic effect. In a different study, biosurfactant extracted from corn steep liquor was used to biosynthesize Ag- and Au-NPs. The bioreduction process was completed in one step under a controlled temperature at 60 ◦C and resulted in a mixture of nanospheres and nanoplates. Biosurfactants were essential for the bioreduction process and also for stabilization of the produced NPs, which improved the antimicrobial activity of the NPs [118].

Green synthesis of Au-NPs by *Salicornia brachiata* (Sb) plant extract and characterization of the formed NPs revealed that mixing plant extract (50 mL) with 10 mM NaBH<sup>4</sup> was sufficient to yield the purple color that indicated the formation of Sb-Au-NPs [119]. TEM micrographs showed that the size of Sb-Au-NPs was approximately 30 nm, while XRD and EDS data proved that Sb-Au-NPs had a pure crystalline form.

#### 4.2.2. Algae, Microalgae, Cyanobacteria, and Diatoms

Algae, microalgae, and cyanobacteria have emerged as attractive biofabrication machines for many NPs [4,68]. The synthesis and antimicrobial and antioxidant applications of Au- and Ag-NPs produced through the exploitation of cell-free extracts of the microalga *Neodesmus pupukensis* were explored [120]. Zone of inhibition tests showed that Ag-NPs were active against *Pseudomonas* sp. (43 mm), *Escherichia coli* (24.5 mm), *Klebsiella pneumoniae* (27 mm), and *Serratia marcescens* (39 mm). In contrast, Au-NPs only showed activity against *Pseudomonas* sp. (27.5 mm) and *Serratia marcescens* (28.5 mm). Antifungal tests indicated that Ag-NPs had mycelial inhibition of 80.6, 57.1, 79.4, 65.4, and 69.8% against *Aspergillus niger*, *A. fumigatus*, *A. flavus*, *Fusarium solani*, and *Candida albicans*, respectively, while Au-NPs had 79.4, 44.3, 75.4, 54.9, and 66.4% against *A. niger*, *A. fumigatus*, *A. flavus*, *F. solani*, and *C. albicans*, respectively. The free radical scavenging power of Au-NPs and Ag-NPs

was 68.9 and 41.21%, respectively. The authors concluded that Au- and Ag-NPs fabricated by *Neodesmus pupukensis* have significant potential as antimicrobial and antioxidant agents and could be used for various biotechnological applications.

Colin et al. reported an eco-friendly green synthesis method to produce Au-NPs with enhanced biocompatibility [76]. The method used an extract from the alga *Egregia* sp., which naturally contains biomolecules that are important for shell formation around the Au-NPs to improve their biocompatibility. The algae extract functions as the reducing agent and as the stabilizing capping shell for the Au-NPs colloid. The yielded Au-NPs had a diameter of approximately 8 nm with a narrow size distribution.

El-Kassas et al. revealed that the formation and stabilization of Au-NPs using *Corallina officinalis* extract could be attributed to the existence of the hydroxyl functional group of polyphenols and the carbonyl group of proteins [121]. Hamida et al. extracellularly synthesized, for the first time, Ag-NPs using the novel cyanobacterial strain *Desertifilum* IPPAS B-1220 [28]. The green Ag-NPs ranged from 4.5 to 26 nm in size, were spherical, and exhibited potent anticancer and antibacterial activities. Similarly, *Nostoc Bahar M* sp. exhibited a potent reducible activity to fabricate silver nitrate into Ag-NPs at ambient temperature after 24 h under dark conditions [58]. The biogenic Ag-NPs were spherical with an average diameter of 14.9 nm and showed antiproliferative activity against colon cancer cells.

Diatoms are unicellular photosynthetic microalgae that are distinguished by hydrated amorphous silica exoskeletons of different sizes and shapes [122]. The use of live diatoms in biotechnology and their applications in ecological monitoring and biofuel production were reported in several studies [123,124]. The biosynthesis of metal NPs using live diatoms as a reducing agent has been demonstrated [125,126]. Jena et al. reported the formation of Ag-NPs by a light-dependent reaction in an aqueous cell extract of diatom *Amphora* sp. [127]. The aqueous extract of *Amphora* sp. was light yellow, indicating that only yellow pigment was extracted but not the chlorophyll. The aqueous extract was added to the silver nitrate solution for the biosynthesis of Ag-NPs. The reaction mixture started to change color from light yellow to brown within seconds and became red brown within 30 min. Ag-NPs were formed only in light conditions because no color change was observed when the reaction was conducted in dark conditions. UV–Vis spectroscopy of the Ag-NP suspension showed a peak at 413 nm. The authors reported that the increase in peak intensity at 413 nm, which was linked to the time of reaction, confirmed the rise in the number of Ag-NPs in the reaction mixture. TEM analysis revealed that Ag-NPs were polydispersed, spherical, and ranged in size from 5 to 70 nm, with an average particle size of 20–25 nm. XRD spectra revealed four intense diffraction peaks at 2θ values of 38.48◦ , 44◦ , 64.74◦ , and 77.4◦ corresponding to (111), (200), (220), and (311) planes, indicating the crystallinity of Ag-NP. These findings indicated that aqueous extract of *Amphora* sp. diatom was highly effective in reducing Ag ions to formulate scattered Ag-NPs.
