4.501 5.

Review Editor’s Choice

Green Synthesis of Metallic

Nanoparticles: Applications and
Limitations

Pritam Kumar Dikshit, Jatin Kumar, Amit K. Das, Soumi Sadhu, Sunita Sharma, Swati Singh,
Piyush Kumar Gupta and Beom Soo Kim

Special Issue
Recent Advances on Nano-Catalysts for Biological Processes
Edited by
Dr. Pritam Kumar Dikshit and Prof. Dr. Beom Soo Kim

https://doi.org/10.3390/catal11080902
 catalysts
Review
Green Synthesis of Metallic Nanoparticles: Applications
and Limitations
Pritam Kumar Dikshit 1, * , Jatin Kumar 1 , Amit K. Das 1 , Soumi Sadhu 1 , Sunita Sharma 2 , Swati Singh 1 ,
Piyush Kumar Gupta 1 and Beom Soo Kim 3, *

1 Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida 201310,
Uttar Pradesh, India; jatin.kumar1@sharda.ac.in (J.K.); amit.das@sharda.ac.in (A.K.D.);
soumi.sadhu@sharda.ac.in (S.S.); swati.singh1@sharda.ac.in (S.S.); piyush.kumar1@sharda.ac.in (P.K.G.)
2 Department of Biotechnology, School of Engineering and Technology, Sharda University,
Greater Noida 201310, Uttar Pradesh, India; sunita.sharma@sharda.ac.in
3 Department of Chemical Engineering, Chungbuk National University, Cheongju 28644, Korea
* Correspondence: pritam.kumar@sharda.ac.in (P.K.D.); bskim@chungbuk.ac.kr (B.S.K.)

Abstract: The past decade has witnessed a phenomenal rise in nanotechnology research due to
its broad range of applications in diverse fields including food safety, transportation, sustainable
energy, environmental science, catalysis, and medicine. The distinctive properties of nanomaterials
(nano-sized particles in the range of 1 to 100 nm) make them uniquely suitable for such wide range
of functions. The nanoparticles when manufactured using green synthesis methods are especially
desirable being devoid of harsh operating conditions (high temperature and pressure), hazardous
chemicals, or addition of external stabilizing or capping agents. Numerous plants and microorgan-
isms are being experimented upon for an eco–friendly, cost–effective, and biologically safe process





 optimization. This review provides a comprehensive overview on the green synthesis of metallic NPs
using plants and microorganisms, factors affecting the synthesis, and characterization of synthesized
Citation: Dikshit, P.K.; Kumar, J.;
NPs. The potential applications of metal NPs in various sectors have also been highlighted along
Das, A.K.; Sadhu, S.; Sharma, S.;
with the major challenges involved with respect to toxicity and translational research.
Singh, S.; Gupta, P.K.; Kim, B.S. Green
Synthesis of Metallic Nanoparticles:
Applications and Limitations.
Keywords: green synthesis; metal nanoparticles; wastewater treatment; agriculture; food application
Catalysts 2021, 11, 902. https://
doi.org/10.3390/catal11080902

Academic Editor: Pierluigi Barbaro 1. Introduction

During the last two decades, nanotechnology has taken massive leaps to become
Received: 20 June 2021 one of the most researched and booming fields due to its applications in various fields of
Accepted: 23 July 2021 human welfare. Nanoparticles (NPs) are naturally occurring or engineered extremely small
Published: 26 July 2021
sized particles in the range of 1 to 100 nm. They exhibit unique and valuable physical and
chemical properties. At nanoscale, particles display better catalytic, magnetic, electrical,
Publisher’s Note: MDPI stays neutral
mechanical, optical, chemical, and biological properties. Due to high surface to volume
with regard to jurisdictional claims in
ratio, NPs show higher reactivity, mobility, dissolution properties, and strength [1]. NPs
published maps and institutional affil-
are thought to have been present on earth since its origin in the form of soil, water, volcanic
iations.
dust, and minerals. Besides their natural origin, humans have also started synthesizing
NPs through various methods [2]. NPs and their derived nanomaterials are finding wide
application in various sectors such as food, agriculture, cosmetics, medicines, etc. Applica-
tion of NPs in food sector involves food processing and preservation (nanopreservatives,
Copyright: © 2021 by the authors. toxin detection, nanoencapsulated food additives, etc.) and food packaging (nanocoatings,
Licensee MDPI, Basel, Switzerland. nanosensors, nanocomposites, edible coating NPs, etc.) In agriculture, nanotechnology is
This article is an open access article
being utilized for the production of nano-fertilizers, pesticides, herbicides, and sensors.
distributed under the terms and
In medicine, nanotechnology involves production of various antibacterial, antifungal, an-
conditions of the Creative Commons
tiplasmodial, anti–inflammatory, anticancer, antiviral, antidiabetic, and antioxidant agents.
Attribution (CC BY) license (https://
Nanotechnology is also useful for the early detection of life-threatening diseases such
creativecommons.org/licenses/by/
as cancer. Besides, NPs have also been used for bioremediation due to their capacity to
4.0/).

Catalysts 2021, 11, 902. https://doi.org/10.3390/catal11080902 https://www.mdpi.com/journal/catalysts

Catalysts 2021, 11, 902 2 of 35

degrade various pollutants such as organic dyes and chemicals. Given the diverse scope of
nanomaterials, different countries are investing in nanotechnology with USA and China
emerging at the top. In 2019, the global market of different nano products was more than
8 billion US dollars, which is expected to show annual growth rate of around 13% by 2027.
Depending on their chemical composition, four major classes of NPs are described,
such as carbon-based (nanotubes and nanofibers of carbon, etc.), metal and metal ox-
ide based (Ag, Cu, etc.), bio-organic based (liposomes, micelles, etc.), and composite
based [3]. NPs can also be classified as organic and inorganic in nature [4]. Organic NPs
are biodegradable in nature and include polymeric NPs, lipid based nanocarriers, lipo-
somes, carbon-based nanomaterials, and solid lipid NPs, while inorganic NPs are based on
inorganic materials comprising of metals and metal oxides such as silver oxide, zinc oxide,
etc. Among all the synthesized NPs, silver NPs (Ag NPs) are the most widely employed,
showing their dominance in various consumer products (more than 25%) [5]. AgNPs are
majorly used as antibacterial, antifungal, and antiviral agents. With each passing year,
novel varieties of NPs are being developed using state-of-art technology having diverse
applications in various sectors.
The synthesis of NPs can be carried out following two different approaches, viz.,
(i) top-down approach, and (ii) bottom-up approach [6,7]. Furthermore, three different
strategies such as physical, chemical, and biological methods are adopted for the synthesis
of NPs. A schematic representation of various methods adopted for NPs synthesis and its
applications is depicted in Figure 1.

Figure 1. Schematic representation of various methods adopted for NP synthesis and its applications.

The physical methods belong to the category of top-down approach, while the
chemical and biological methods follow the bottom-up approach for the synthesis NPs.
Evaporation-condensation, electrolysis, diffusion, laser ablation, sputter deposition, pyrol-
ysis, plasma arcing, and high energy ball milling are some of the most common physical
methods used for the synthesis of NPs [8]. However, low production rate, expensive opera-
tions, and high energy consumption are the major limitations of these processes. Conversely,
chemical synthesis methods that include chemical reduction, micro-emulsion/colloidal,
electrochemical, and thermal decomposition are the conventional and most widely used
methods for the synthesis of metallic NPs. The chemical reduction of NPs from their respec-
tive metal salt precursors by adding particular reducing agents is one of the most widely
used methods for NPs chemical synthesis due to easy operational and equipment require-
ment. Several reducing agents, such as sodium borohydride (NaBH4 ) [9], potassium bitar-
trate [10], formaldehyde [11], methoxypolyethylene glycol [12], hydrazine [13], etc., and
stabilizing agents like dodecyl benzyl sulfate [14] and polyvinyl pyrrolidone [15] have been
explored during synthesis. The chemical methods are economical for large-scale production;
however, the use of toxic chemicals and production of harmful by-products cause environ-
mental damage, thereby limiting its clinical and biomedical applications [16,17]. Hence,
Catalysts 2021, 11, 902 3 of 35

there is an increased demand for reliable, nontoxic, high-yielding, and eco-sustainable

techniques for metallic NPs that can replace the conventional methods. The biological
synthesis methods, therefore, provide an attractive alternative to the physicochemical
synthesis methods.
The present article provides a critical overview on the synthesis of metallic NPs using
biological methods and several factors affecting the preparation process. The applications
of NPs in various sectors, such as medicine, wastewater treatment, agricultural sectors,
etc., have been discussed in detail. Current challenges highlighting the toxic effects of
NPs and future perspectives in each section gives us a comprehensive way forward in
the near future.

2. Biological Synthesis of NPs

The biological synthesis of NPs can be carried out using a vast array of resources such
as plants and plant products, algae, fungi, yeast, bacteria, and viruses. The synthesis of
NPs is initiated by the mixing of noble metal salt precursors with biomaterials [18]. The
presence of various compounds, such as proteins, alkaloids, flavonoids, reducing sugars,
polyphenols, etc., in the biomaterials act as reducing and capping agents for the synthesis
of NPs from its metal salt precursors [19]. The reduction of metal salt precursor to its
successive NPs can be initially confirmed by visualizing the color change of the colloidal
solution. Several studies reported the synthesis of Ag, Au, Cu, Pt, Cd, Pt, Pd, Ru, Rh, etc.
using various biological agents in the recent past.

2.1. Plant-Mediated Synthesis of NPs

Figure 2 shows the Scopus search (with keywords “metal nanoparticles” and “plant
extract”) results of the number of research published from last 10 years on biological
synthesis of NPs. An increase in the number of research publications was observed with
each year and approximately 468 publications reported in the year 2020. These data further
corroborate that the research interest in the area of biological NPs using plant extract is
increasing significantly every year.

Figure 2. Number of research publications on biological synthesis of metallic NPs from last 10 years.
Source: Scopus. * Number of publications reported as on 10 May 2021.

Synthesis of a wide range of metallic NPs has been reported using various plants [6,20,21].
Plant mediated synthesis of NPs can be achieved by three different methods, viz., (i) in-
tracellularly (inside the plant), (ii) extracellularly (using plant extracts), and (iii) using
individual phytochemicals. Several plants have the capability of metal accumulation and
successive conversion of these accumulated metals to NPs intracellularly. The presence of
several biomolecules such as amino acids, alkaloids, aldehydes, flavones, ketones, proteins,
phenolics, polysaccharides, saponins, tannins, terpenoids, and vitamins in the plant plays
a key role in the reduction of metals [22]. The variation in the size, shape, and properties of
Catalysts 2021, 11, 902 4 of 35

accumulated NPs are observed due to the variation in stabilizing and reducing potential
of biomolecules present in the plant. The formation of gold NPs inside the living plant,
alfalfa was reported by Gardea-Torresdey et al. [23] when the plants were grown in AuCl4
rich environment. In a similar kind of study, Bali and Harris [24] observed the ability of
Medicago sativa and Brassica juncea plants to accumulate Au NPs from aqueous solutions of
KAuCl4 . The NPs were majorly located in the xylem parenchyma cells while some were
also accumulated throughout the epidermis, cortex, and vascular tissue.
However, for the past several years, most of the works have been focused on using
the inactive part of the plants either in powder form or as an extract for the synthesis
of NPs [25]. Table 1 summarizes the green synthesis of various metal NPs using plants.
Various parts of plants such as leaves, steam, flower, fruit, root, latex, seed, and seed coat
are being exploited for the synthesis of metallic NPs.

Table 1. Summary on synthesis of metallic nanoparticles by various plant species.

Nanoparticles Plant Species Experimental Conditions Shape and Size References

Silver (Ag) Acalypha indica Linn Temperature: 27 ◦ C; pH: 7.0; duration: 30 min Spherical; 20–30 nm [26]
Temperature: 20–100 ◦ C; pH: 2.0–10.0;
Chenopodium album leaf Spherical; 10–30 nm [27]
duration: 15 min
Hibiscus rosa sinensis leaf pH: 7.2–8.5 Spherical; 13 nm [28]
Calendula officinalis seed Temperature: 30 and 60 ◦ C; pH: 3.0–9.0 Spherical; 7.5 nm [29]
Allophylus cobbe leaf Temperature: 60 ◦ C; pH: 8.0; duration: 6 h Spherical; 2–10 nm [30]
Cissusquadrangularis leaf Duration: 60 min Spherical and cuboidal [31]
Piper nigrum, Ziziphus Spina—Christi
Temperature: ambient; duration: 1 h Spherical; 8−35 nm [32]
and Eucalyptus globulus leaves
Phyllanthus emblica fruit Temperature: 65 ◦ C; duration: 2 h Spherical; 16.29 nm [33]
Blumea eriantha DC Temperature: ambient; duration: 2–3 h Spherical; 50 nm [34]
Brillantaisia patula, Crossopteryx febrifuga
Temperature: 70 ◦ C; duration: 24 h Spherical; 45–110 nm [35]
and Senna siamea leaf
Ocimum tenuiflorum leaf Temperature: ambient; duration: 10 min Spherical and ovoid; 7–15 nm [36]
Annona squamosa leaf Temperature: ambient Spherical; 20–100 nm [37]
Aloe leaf Temperature: ambient; duration: 20 min Spherical; 20 nm [38]
Artocarpus heterophyllus Lam.
Temperature: 121 ◦ C; duration: 5 min Irregular; 3–25 nm [39]
Seed
Trigonella foenum graecum seed Duration: 5 min Spherical; 17 nm [40]
Andrographis paniculata Temperature: 30–95 ◦ C Spherical; 13–27 nm [41]
Temperature: 20–60 ◦ C; pH: 4.5–10.0;
Podophyllum hexandrum leaf Spherical; 12–40 nm [42]
duration: 30–150 min
Syzygium cumini fruit Temperature: ambient; pH: 7.0–9.0; duration: 2 h Spherical; 5–20 nm [43]
Crassocephalum rubens leaf Temperature: 50 ◦ C; duration: 20 min Spherical and hexagonal; 15–25 nm [44]
Rectangular and triangular;
Gold (Au) Cassia fistula stem bark Temperature: ambient [45]
55.2–98.4 nm
Crassocephalum rubens leaf Temperature: 50 ◦ C; duration: 10 min Spherical; 10–20 nm [44]
Simarouba glauca leaf Duration: 15 min Spherical and prism; <10 nm [46]
Temperature: 30–100 ◦ C; pH:2.0–12.0; duration: Spherical, polygonal, rod and
Hygrophila spinosa [47]
15–60 min triangular; 68 nm
Croton Caudatus Geisel leaf Temperature: ambient Spherical; 20–50 nm [48]
Triangular, hexagonal, and spherical;
Moringa oleifera flower Temperature: ambient; duration: 60 min [49]
5 nm
Temperature: 25–50 ◦ C; pH: 2.0–10.00;
Illicium verum Triangular and hexagonal; 20–50 nm [50]
duration: 15 min
Terminalia arjuna leaf Temperature: ambient; duration: 15 min Spherical; 20–50 nm [51]
Temperature: 37 and 50 ◦ C; pH: 7.4;
Zingiber officinale Spherical; 5–10 nm [52]
duration: 20 min
Spherical, triangular, and hexagonal;
Rosa hybrida petal Temperature: ambient; duration: 5 min [53]
10 nm.
Triangular, pentagonal, and
Terminalia chebula seed Temperature: ambient; duration: 20 s [54]
spherical; 6–60 nm
Temperature: 30–60 ◦ C; pH: 5.0– 13.0;
Eucommia ulmoides bark Spherical [55]
duration: 30 min
Acorus calamus rhizome Temperature: ambient; pH: 4.0–9.2 Spherical; 10 nm [56]
Curcuma pseudomontana root Temperature: ambient; duration: 30 min Spherical shape; 20 nm [57]
Citrus limon, Citrus reticulata and Citrus
Temperature: ambient; duration: 10 min Spherical and triangular; 15–80 nm [58]
sinensis
Catalysts 2021, 11, 902 5 of 35

Table 1. Cont.

Nanoparticles Plant Species Experimental Conditions Shape and Size References

Palladium Hippophae rhamnoides
Temperature: 80 ◦ C; duration: 25 min 2.5–14 nm [59]
(Pd) Linn leaf
Cinnamom zeylanicum bark extract Temperature: 30 ◦ C; pH: 1.0–11.0; duration: 72 h Spherical; 15–20 nm [60]
Temperature: 40–100 ◦ C; pH: 2.0–5.0;
Banana peel extract 50 nm [61]
duration: 3 min
Quasi–spherical and irregular;
Cinnamomum camphora leaf Temperature: ambient; duration: 12 h [62]
3.6–9.9 nm
Catharanthus roseus leaf Temperature: 60 ◦ C; duration: 2 h Spherical; 38 nm [63]
Terminalia chebula fruit Temperature: ambient; duration: 40 min - [64]
Rosmarinus officinalis Temperature: ambient; duration: 24 h Semi–spherical; 15–90 nm [65]
Anogeissus latifolia Duration: 30 min Spherical; 2.3–7.5 nm [66]
Rod; diameter—20 nm,
Daucus carota leaves - [67]
length—38–48 nm
Camellia sinensis leaves Temperature: 100 ◦ C; duration: 1 h Spherical; 5–8 nm [68]
Platinum
Anacardium occidentale leaf Temperature: ambient; pH: 6.0–8.0 Irregular rod shaped [69]
(Pt)
Cacumen platycladi Temperature: 30–90 ◦ C; duration: 25 h Spherical; 2–2.9 nm [70]
Asparagus racemosus root Duration: 5 min 1.0–6.0 nm [71]
Diopyros kaki leaf Temperature: 25–95 ◦ C Spherical and plate; 2–20 nm [72]
Ocimum sanctum leaf Temperature: 100 ◦ C; duration: 1 h Rectangular and triangular; 23 nm [73]
Copper Spherical and non–regular;
Mulberry fruit (Morus alba L.) Temperature: ambient; duration: 5 h [74]
(Cu) 50–200 nm
Crotalaria candicans leaf - Spherical; 30 nm [75]
Ziziphus spinachristi fruit Temperature: 80 ◦ C Spherical; 5–20 nm [76]
Clove (Syzygium aromaticum) buds ◦
Temperature: 30 C; duration: 15 min Spherical; 15–20 nm [77]
Iron (Fe) Tea leaves extract Temperature: 80 ◦ C; duration: 3 h 30–100 nm [78]
Moringa oleifera seeds Temperature: ambient; duration: 30 min Spherical; 2.6–6.2 nm [79]
Trigonella foenum–graecum seed Temperature: 30 ◦ C; duration: 5 min 7–14 nm [80]
Selenium Monodispersed and spherical;
Ocimum tenuiflorum Temperature: ambient; duration: 75 h [81]
(Se) 15–20 nm
Murraya koenigii - Spherical; 50–150 nm [82]
Zinziber officinale fruit Temperature: ambient; pH: 9.0; duration: 75 h Spherical; 100–150 nm [83]
Nickel (Ni) Calotropis gigantea leaves Temperature: 80 ◦ C; pH: 12.0; duration: 90 min 60 nm [84]
Desmodium gangeticum roots Temperature: 80 ◦ C; duration: 45 min - [85]

In general, the synthesis of NPs is carried out by mixing the plant biomass/extract with
a metal salt solution at a desired temperature and pH. The primary confirmation of NPs
synthesis can be checked by looking at the color change of the solution. The experimental
procedure for the synthesis of NPs using plant biomass is depicted in Figure 3.

Figure 3. Schematic representation for plant-mediated biosynthesis of nanoparticles.

The plant extracts are prepared by using different methods such as hot extraction,
cold extraction, and using Soxhlet apparatus, which were later used in NPs synthesis. This
method of synthesis of NPs is more suitable in comparison to the intracellular method
due to easy scale-up and downstream processing. Additionally, this method is renewable,
non-toxic, biocompatible, and eco-friendly. Due to their biocompatible nature, these
NPs are known to have various biological applications. The synthesis of metal NPs is
Catalysts 2021, 11, 902 6 of 35

initiated by adding the plant extract to the metal precursor solution containing the salts
of respective metals. Metal precursor solutions such as AgNO3 , HAuCl4 , PdCl2 , H2 PtCl6 ,
Cu(NO3 )2 ·3H2 O, FeCl3 ·6H2 O, Na2 SeO3 , and (NiNO3 )2 ·6H2 O are commonly used for the
synthesis of Ag, Au, Pt, Cu, Fe, Se, and Ni NPs. The synthesis of metal NPs using plant
extract mainly occurs in three stages. In the first stage, the reduction of metal ions (M+ or
M2+ ) to metal atoms (M0 ) and successive nucleation of the reduced metal atoms occurs.
While in the second stage, the coalescence of small adjacent NPs into larger size particles
occurs with simultaneous increase in thermodynamic stability. At the final stage, the
termination of the process takes place while giving the final shape to the NPs [86,87]. The
presence of various active biomolecules in the plant extract plays an important role in the
reduction and stabilization of metal ions in the solution. However, due to the presence of
a large number of phytochemicals in the plant extract, it is difficult to ascertain the exact
reducing and stabilizing agents for NPs synthesis.
Salih et al. [32] used plant extract derived from leaves of three different plants, viz.,
Piper nigrum, Ziziphus Spina—Christi and Eucalyptus globulus, for the synthesis of AgNPs.
The average particle size distribution was in the range of 8–35 nm and decreased with
the increase in concentration of plant extract. In a similar kind of study, Dhar et al. [33]
reported the synthesis of AgNPs using fruit extract of Phyllanthus emblica. The fabricated
AgNPs were spherical with an average size of 60–80 nm. The extraction methods also
play an important role which influences the antioxidant properties of Ag and Au NPs
synthesized using leaves extract of Crassocephalum rubens [44]. Figure 4 depicts the size,
shape, and morphological features of C. rubens synthesized AgNPs and AuNPs. The SEM
and TEM images revealed spherical and hexagonal shapes of AgNPs with size 10–15 nm,
whereas the size of AuNPs was in the range of 10–20 nm with spherical shape.

Figure 4. Characterization of AgNPs and AuNPs synthesized from Crassocephalum rubens leaf extract.
TEM images of (A) AgNPs and (B) AuNPs; SEM images of (C) AgNPs and (D) AuNPs. Reprinted
with permission from Reference [44].Copyright 2021 Elsevier.

The biosynthesized Au NPs using aqueous extract of Hygrophila spinosa exhibited

enhanced cytotoxicity against various cancer cell lines compared to H. spinosa aqueous
extract [47]. These green synthesized AuNPs with antioxidant and cytotoxic properties
could provide a new direction for the development of nanomedicine. In addition to Au
Catalysts 2021, 11, 902 7 of 35

and Ag NPs, Pd NPs synthesized using Rosmarinus officinalis leaves extract demonstrated
notable antimicrobial and antifungal activity against different bacteria and fungi [65].
Besides leaves, other plant parts such as root, fruit, flower, petal, seed, peel, bark etc. are
used in the biosynthesis of different NPs [40,49,53,55,61,71,80]. Several previous studies
tried to explain the mechanisms behind the antimicrobial effect of metallic NPs [88–90].
These metal ions could strongly interact with the negatively charged bacterial cell wall
leading to its rupture. The other mechanism states distortion of the helical structure of
bacterial DNA due to the interaction of NPs and interruption of internal and external
cellular mechanisms.

2.2. Microbial Synthesis of NPs

In addition to the plant-mediated synthesis, several microorganisms such as bacteria,
fungi, actinomycetes, and viruses are also reported to synthesize various metal NPs. The
interaction between metals and microorganisms have been exploited in the past for various
biological applications such as biomineralization, bioremediation, bioleaching, and biocor-
rosion [91]. However, recently microbial synthesis of NPs has emerged as a promising field
of research due to certain advantages compared to other methods. The NPs are synthesized
either intracellularly or extracellularly depending on the type of microorganisms [92].
Previous reports on the synthesis of metal NPs using various microorganisms have been
summarized in Table 2.

Table 2. Summary on synthesis of metallic nanoparticles by microorganisms.

Microorganisms Nanoparticles Shape and Size References

Bacteria

Bacillus subtilis Ag Spherical; 3–20 nm [93]

Pseudomonas stutzeri Ag Triangular; 200 nm [94]
Bacillus licheniformis Ag 40 nm [95]
Ochrobactrum anhtropi Ag Spherical; 38–85 nm [96]
Pantoea ananatis Ag Spherical; 8.06–91.31 nm [97]
Actinobacter Ag Spherical; 13.2 nm [98]
Pseudomonas aeruginosa Au 15–30 nm [99]
Rhodopseudomonas capsulata Au Spherical; 10–20 nm [100]
Escherichia coli DH5α Au Spherical, triangles, and quasi–hexagons; 25 nm [101]
Bacillus subtilis Au Spherical, 20–25 nm [102]
Mycobacterium sp. Au Spherical; 5–55 nm [103]
Shewanella loihica Pt 1–10 nm [104]
Shewanella oneidensis MR–1 Pt 2.83–61.03 nm [105]
Jeotgalicoccus coquinae ZC15 Pt Spherical; 5.74 nm [106]
Shewanella loihica Pd 1–12 nm [104]
Shewanella oneidensis MR–1 Pd 10–100 nm [107]
Lysinibacillus sp. ZYM-1 Se Cubic; 100–200 nm [108]
Bacillus subtilis Se Spherical; 50–400 nm [109]
Lactobacillus acidophilus Se Spherical; 2–15 nm [110]

Fungi

Rhizopus stolonifer Ag Spherical; 2.86 nm [111]

Candida glabrata Ag Spherical; 2–15 nm [112]
Trametes trogii Ag Spherical and rod; 5–65 nm [113]
Trichoderma longibrachiatum Ag Spherical; 10 nm [114]
Fusarium oxysporum Ag Spherical; 21.3–37 nm [115]
Aspergillus terreus Ag Spherical; 7–23 nm [116]
Ganoderma sessiliforme Ag Spherical; 45 nm [117]

Candida albicans ATCC 10231 Ag Spherical; 10–20 nm [118]

Cladosporium cladosporioides Au 60 nm [119]
Trichoderma harzianum Au Spherical; 26–34 nm [120]
Pleurotus ostreatus Au Spherical; 10–30 nm [121]
Aspergillus sp. Au Spherical; 4–29 nm [122]
Rhizopus oryzae Au Spherical and flower like structure; 16–43 nm [123]
Penicillium chrysogenum Pt Spherical; 5–40 nm [124]
Fusarium oxysporum f. sp. lycopersici Pt Triangle, hexagons, square, and rectangles; 10–50 nm [125]
Fusarium oxysporum Si Quasi–spherical; 5–15 nm [126]
Fusarium oxysporum Ti Spherical; 6–13 nm [126]

Yeast

Rhodotorula sp. ATL72 Ag Spherical and oval; 8–21 nm [127]

Saccharomyces cerevisiae Ag Spherical; 2–20 nm [128]
Cryptococcus laurentii Ag 35–400 nm [129]
Rhodotorula glutinis Ag 15–220 nm [129]
Rhodotorula glutinis Ag Spherical; 15.5 nm [130]
Saccharomyces cerevisiae Au Triangle, truncated triangle, and hexagon [131]
Magnusiomyces ingens LHF1 Au Spherical and pseudo–spherical; 20–28 nm [132]
Saccharomyces cerevisiae Pd Hexagonal; 32 nm [133]
Magnusiomyces ingens LHF1 Se Spherical and quasi–spherical; 70–90 nm [134]

Compared to other microorganisms, bacteria are preferred for the synthesis of NPs due
to their easy maintenance, high yield, and low purification cost. In recent years, cell-free
extract of endophytic bacterium, Pantoea ananatis, was used for the synthesis of AgNPs [97].
These synthesized spherical shaped NPs with an average size ranging from 8.06–91.32 nm
exhibited significant antimicrobial activity against various pathogenic microorganisms.
Catalysts 2021, 11, 902 8 of 35

In a similar kind of study, Wypij et al. [98] reported maximum antimicrobial activity of
AgNPs synthesized by using acidophilic actinobacterial strain against E. coli, followed
by B. subtilis and S. aureus. Moreover, bacteria species belonging to Pseudomonas stutzeri,
Bacillus licheniformis, Ochrobactrum anhtropi, Bacillus subtilis, and Actinobacter were used for
the synthesis of AgNPs (Table 2). Gold NPs were synthesized by Pseudomonas aeruginosa,
Rhodopseudomonas capsulate, Bacillus subtilis, Escherichia coli DH5α, Mycobacterium sp., etc.
A larger amount of Actinobacter in the medium led to the formation of smaller size with
uniformly distributed spherical AuNPs [135]. Intercellular and extracellular components
of microorganisms play an important role in the synthesis of AuNPs [136]. Intercellular
components such as reducing sugar, fatty acids, and enzymes and extracellular such as
exopolysaccharides help in the reduction of AuNPs [136].
In addition to bacteria, the synthesis of NPs using fungi has received increasing
attention due to various advantages such as easy scale-up and downstream processing,
economic feasibility, and increased surface region due to the presence of mycelia. Different
species of fungi such as Rhizopus stolonifera, Candida glabrata, Trametes trogii, Trichoderma
longibrachiatum, Aspergillus terreus, Fusarium oxysporum, Ganoderma sessiliforme, Candida
albicans ATCC 10231, Cladosporium cladosporioides, Trichoderma harzianum, Pleurotus ostreatus,
Aspergillus sp., Rhizopus oryzae, etc. are used in silver and gold NPs synthesis. Other species
of fungi such as Penicillium chrysogenum and Fusarium oxysporum are used for the synthesis
of Pt, Si, and Ti NPs.
Among the eukaryotic organism, yeast has been used for the synthesis of NPs like
Ag, Au, Pd, Se, etc. Soliman et al. [127] synthesized AgNPs using pink yeast Rhodotorula sp.,
and the characterization revealed the NPs to be spherical and oval in shape with
8.8–21.4 nm size. These biosynthesized AgNPs exhibited significant antimicrobial activity
with complete inhibition to wide range of bacteria (i.e., both Gram positive and Gram
negative) as well as fungi. Similarly, the antifungal activity of biosynthesized AgNPs
using two yeasts: Rhodotorula glutinis and Cryptococcus laurentii was evaluated against the
phytopathogenic fungi [129]. The results of this study revealed that the antifungal activity
of AgNPs from R. glutinis was higher than that from the ones prepared from C. laurentii. In
another study, the morphology and size of AuNPs were controlled by varying the pH of the
medium containing yeast [131]. In this method, various morphologies of gold nanoplates
such as triangle, truncated triangle, and hexagonal nanoplates with uniform size were
synthesized successfully (Figure 5).

Figure 5. SEM images of gold nanoplates synthesized using yeast extract at different pH conditions. (A) Au nanoplates at
low pH without NaOH, (B) small Au nanoplates synthesized at high pH. Reprinted with permission from Reference [131].
Copyright 2016 Springer Nature.
Catalysts 2021, 11, 902 9 of 35

Similarly, the palladium (Pd) and selenium (Se) NPs are synthesized using aqueous
extract of Saccharomyces cerevisiae and cell-free extracts of Magnusiomyces ingens yeast,
respectively [133,134].
The NPs are formed during the microbial synthesis process is due to the oxida-
tion/reduction of metallic ions by secreted biomolecules by microbial cells such as en-
zymes, sugars, carbohydrates, proteins, etc. [137]. However, a complete understanding
of microbial NP synthesis is still unknown as the routes for NPs synthesis varies for each
kind of microorganisms. The reduction of silver for the synthesis of extracellular and
intracellular AgNPs by bacteria is mainly achieved by the action of deoxyribonucleic acid
(DNA) or sulfur-containing proteins, whereas in the case of fungi, the process is carried
out by nitrate-dependent reductase or carboxylic group [138]. The extracellular synthesis
method is preferable due to easy and simpler purification steps. In contrast, the intra-
cellular NPs synthesis method is challenging and expensive due to the involvement of
additional separation and purification processes. Fungal-mediated synthesis of NPs holds
additional advantages compared to algae or bacteria in terms of easier and simpler biomass
handling and downstream processes along with the secretion of large amount protein
that further increases the productivity by several folds. However, the microorganism
mediated NPs synthesis process is extremely intricate and difficult due to the preparation
of inoculum and growth media, isolation of strain, and maintenance of culture medium
and operation conditions (pH, temperature, agitation). Conversely, the use of plant extracts
or broths is simple and convenient, devoid of the complex methods of cell culture and
maintenance. The time required to achieve a complete reduction of NPs using microor-
ganisms is usually 24 to 120 h, while the reduction time is much less in using plant extract
ranging from few hours to 48 h [138]. The reduction rate using the plant is much faster
than microorganisms and in close agreement with the physical and chemical methods. The
use of microorganisms for large-scale biosynthesis of NPs lacks feasibility compared with
plants, which require less time for reaction completion. As reported in the earlier studies,
the plant-mediated biosynthesis of AgNPs demonstrate better production rate, size, and
morphological characteristics compared to other available biological techniques [138].
Hence, the plant-mediated synthesis of NPs proves to be a sustainable alternative
not only to the other biological techniques but also to other synthesis methods such as
physical and chemical. However, in-depth studies are required to understand the detailed
mechanisms of action and to achieve better control over size, morphology, and production
rate for making a plant-mediated synthesis method at par with chemical methods.

2.3. Factors Affecting NPs Synthesis

Adjustment of shape and size of metal NPs further enhances their functionality
for various applications. The morphological parameters of NPs can be manipulated by
changing various experimental parameters such as reaction time, reactant concentration,
pH, temperature, aeration, salt concentration, etc. [139]. Precise control of these parameters
can play a critical role during the optimization of metal NPs synthesis via the biological
route. The size and shape of NPs can be controlled by varying the pH of the medium,
while the acid pH leads to the formation of large-sized NPs [140,141]. During synthesis of
Au NPs using oat (Avena sativa) biomass, Armendariz et al. [142] observed smaller sized
gold NPs at pH 3.0 and 4.0 in comparison to the synthesized NPs at pH 2.0. This is due to
the better accessibility of functional groups present in the extract for nucleation at higher
pH compared to the presence of fewer groups at a lower pH range. In addition to pH, the
concentration of biomolecules in the extract also affects the size and shape of synthesized
NPs. Increase in the concentrations of Aloe vera leaf extract resulted in the synthesis of
higher amount of spherical gold NPs instead of triangular which is due to the presence of
carbonyl compounds in the extract [143]. In addition, the size of the NPs was modulated in
the range of 50 to 350 nm by varying the extract concentration in the solution. The duration
of reaction also plays a crucial role in the reduction of NPs and their size, which is primarily
Catalysts 2021, 11, 902 10 of 35

confirmed by rapid change in color of the reaction mixture. This duration can range from
few minutes to few days.
A change in particle size of silver NPs was observed in the range of 10–35 nm by
increasing the reaction time from 30 min to 4 h using Azadirachta indica leaf extract. Fur-
thermore, the reaction temperature is one of the important parameters in the biological
synthesis of NPs which also determines the shape, size, and yield of NPs. The average size
of silver NPs decreased from 35 to 10 nm with the increase in reaction temperature from
25 to 60 ◦ C using Citrus sinensis (sweet orange) peel extract [144].

2.4. Characterization of NPs

NPs have attracted significant attention of researchers due to their unique physical,
chemical, and mechanical properties. Therefore, the physicochemical characterization of
synthesized NPs is critically important before its application in various sectors. Analyzing
various characteristics such as size, shape, surface morphology, surface area, structure,
stability, elemental and mineral decomposition, homogeneity, intensity, etc. will provide
important information about the NPs, which subsequently determined their end-use ap-
plications. Additionally, the electrical and thermal conductivity and purity of NPs can
also be obtained by using these techniques. Size and shape of the synthesized NPs are
mainly analyzed using X-Ray Diffraction (XRD), Scanning Electron Microscope (SEM),
Field Emission Scattering Electron Microscopy (FESEM), Transmission Electron Microscopy
(TEM), High-Resolution Transmission Electron Microscopy (HRTEM), Atomic Force Mi-
croscopy (AFM), Dynamic Light Scattering (DLS), Condensation Particle Counter (CPC),
Photon Correlation Spectroscopy (PCS), etc. Among these techniques, XRD, SEM, and
TEM are most commonly used for this purpose. Further, SEM, TEM, AFM, etc. are used
for studying the surface morphology. Superconducting Quantum Interference Device
(SQUID), Vibrating Sample Magnetometer (VSM), Electron paramagnetic resonance (EPR),
etc. are used for the determination of magnetic properties of NPs. More details about NPs
characterization techniques are reviewed by previous authors [145,146].

3. Potential Applications of Metal NPs

These synthesized metallic NPs offer a diverse platform for various applications. Some
of the most important applications of these metallic NPs are summarized in this section.

3.1. Agriculture
Nanotechnology has proven its potential to benefit the agriculture sector by finding
solutions to agricultural and environmental problems in order to increase food production
and security [147].

3.1.1. Effect of Nano Material on Plants

Seed germination and later growth phases benefit from nano-growth stimulants [148,149].
Owing to the small size and large surface area of nanoparticles, these particles are able
to seep into the seed pores eventually activating the phytohormones required for seed
growth and germination [150,151]. For example, the use nano-TiO2 and nano-SiO2 on
soybean seedlings improved nitrate reductase activity thereby enhancing seed germination.
However, combining both nanomaterials (NMs) was more advantageous [152]. Seed
treatment with 0.25 percent TiO2 improved nitrogen assimilation and photosynthesis
rate in spinach (Spinacia oleracea L.), resulting in better growth [153,154]. Watermelon
(Citrullus lanatus) seed soaking in Fe2 O3 NPs improved germination and initiated plant
development and fruiting behavior [155]. The use of low-concentration SiO2 NPs on
tomato seeds improved germination [156]. Nanomaterials can be used in a variety of ways
in in-vitro cultivation. Zn as a ZnO nanomaterial resulted in an increased calli growth
and physiological parameters in tobacco (Nicotiana tabacum L.). Although nanoparticles
have been widely utilized for promoting plant growth [149,157], NM application may also
be phytotoxic [158]. The positive and negative effects, however, are dependent on the
Catalysts 2021, 11, 902 11 of 35

dose, size, time, exposure, and make (synthetic/biological) of the NM [158,159]. Over
the past years, application of chemically or physically synthesized NM have proven to be
stimulant of plant growth; however, they pose a greater threat to ecology and environment
(by seeping through the soil) [160]. Utility of green/biological NM in agriculture has
therefore been advantageous due to their safety and feasibility [161]. An increased usage of
biologically synthesized Au, Ag, Ti, Ca, N, Fe nanoparticles either in form of nanofertilizers
or nanopesticides has been employed [161–165].

3.1.2. Application of Nanomaterials in the Field of Agriculture

Over the recent years, application of nanomaterials to boost agriculture has broadly
been in two major forms, either as nanofertilizers, to enhance the agricultural productivity
or in the form of nanopesticides, to eradicate the pest/pathogens/weeds hampering
the growth of crop plants. In this section, we will be discussing the applicability of
nanofertilizers and nanopesticides, in current agricultural practices.

Nanofertilizers
Huge increase in agricultural yields, particularly grain yields, has played an important
role in providing the world’s food demands over the last five decades. In this context,
increased usage of chemical fertilizers acts as one of the key contributors to increased crop
productivity. Although, use of chemical fertilizers has increased productivity of crops, their
poor use efficiency due to volatilization and leaching has led to its excessive usage [147].
On the contrary, nanofertilizers are compounds that are applied in smaller amounts and
can enhance the effect of fertilizers [166]. Enhancing the effect of fertilizers on plants is
usually done by governing the fertilizers in nano form which results in controlled nutrient
release, eventually minimizing the risk of environmental damage [167]. With the recent
advancement in the field of nanobiotechnology, nanofertilizers can be utilized as intelligent
fertilizers which are able to release desired amount of nutrients just when and where they
are needed by plants, thereby limiting the conversion of excess fertilizers to gaseous forms
or leaking downstream [168].
To date, various NPs have been employed in developing fertilizers, some of which
include hydroxyapatite, polyacrylic acid, clay minerals, chitosan, zeolite, and many more.
The small size and large surface area of these NPs give them an advantage over the
conventional fertilizers. For example, strong interactions of hydroxyapatite with urea
lead to release of nitrogen from urea until 60 days as compared to the ammonium nitrate
fertilizer (normal form of urea) which releases nitrogen only until 30 days [169].
Nanofertilizers can be broadly classified into three categories: (1) nanoparticulate
nano fertilizers, (2) micronutrient nanofertilizers, and (3) macronutrient nanofertilizers.
Nanoparticulate nanofertilizers include NPs, such as CNTs, TiO2 , and SiO2 , responsible for
plant growth. In soybean, an amalgamation of TiO2 and SiO2 results in overall increase
in plant growth with increased nitrogen fixation and improved seed germination [152].
As utility of TiO2 nanoparticle in plant growth has been well established, recently several
works have been done for generation of non-toxic, cheap, and environmentally safe green
synthesized TiO2 from plant extracts of Syzgium cumini, Moringa oleifera, Cucurbita pepo,
and Trigonella foenum [170–173]. Micronutrients such as molybdenum (Mo), copper (Cu),
iron (Fe), nickel (Ni), manganese (Mn), and Zinc (Zn) packed in NPs serve as micronutrient
nano fertilizers. A mixture of three micronutrients NPs (ZnO, CuO, and B2 O3) has been
successfully established to ameliorate drought stress in soybean plants [174]. Recent studies
on Zea mays have revealed utility of biologically synthesized micronutrient nanofertilizers
(iron oxide nanorods) in better plant growth as compared to the chemically synthesized
NPs [164]. Similar to micronutrients, macronutrient nanofertilizers are composed of a
combination of macroelements (Mg, K, N, Ca, and P) [167]. Foliar application of Mg and
Fe NPs on Vigna unguiculata, resulted in increased seed weight and photosynthesis ability
thereby resulting in an overall improvement in yield [175]. When compared to crops
administered with conventional fertilizer, phosphatic nanofertilizers have been attributed
Catalysts 2021, 11, 902 12 of 35

a 32 percent rise in growth rate and a 20 percent rise in seed production in soybean
(Glycine max L.) [176]. A recent study on application of green synthesized multinutrient
nanofertilizer (U-NPK) made from calcium phosphate NP doped with potassium and
nitrogen resulted in reduction of 40% of nitrogen requirement of plants when compared
to conventional approach due to slow and gradual release of major micronutrients [165].
Overall, use of nanofertilizer results in reduction in usage of fertilizer amount by allowing
slow-release products.

Nanopesticide
Plants being sessile in nature are prone to various biotic and abiotic stresses such
as pests, pathogens, heat, drought, pollution, etc. These stresses directly or indirectly
affect the total yield of a plant. The ill effect of pathogens on an entire crop leading
to famine like situations has been extensively studied by far, and in order to have a
control over these forms of biotic stress, use of pesticides had been advocated. Utility
of pesticides has been established in eradication of harmful pests and pathogens from
crop field resulting in crop protection [177]. However, it has been found that use of
pesticides leads to deleterious effects on environment and human health. As a result,
numerous pesticides have been prohibited by state or international governments. Thus,
development of effective yet safe pesticides is the need of the hour. Although biopesticides
have emerged as a breakthrough, their use has been limited due to significantly higher cost
of production. However, nanotechnology offers a new and better approach by introduction
of nanopesticides [178].
One of the widely used examples of nanopesticide is nanostructured alumina (NSA).
NSA acts as negatively charged insecticide which interacts with the positively charged bod-
ies of the insects leading to dehydration. The dehydration of insect body results in detach-
ment of insect’s cuticle eventually leading to death [167]. Potent insecticidal activity of sev-
eral other NPs has also been studied over past few years. In the year 2013, Paret et al. [179]
studied the antimicrobial effect of TiO2 and ZnO NPs against X. perforans, casual organism
of tomato spot disease. Role of Imidacloprid (IMI) as an effective systemic insecticide
against several sucking insects such as Martianus dermestoides has been established; in
addition, use of the nano-IMI being more photodegradable increases its effectiveness and
environmental safety over the conventional formulation [180]. Another study found that
nanoformulation of permethrin had a higher absorption than the conventional form against
Aedes aegypti. In a recent study, Zhao et al. investigated the insecticidal activity of Cu NPs
which showed potential upregulation of exogenous microbial protein within plant tissue
enhancing resistance against the bollworm, under the effect of at a low dose of Cu NPs [181].
These findings are encouraging for utility of nanopesticides against various crop pests
which could be an important tool in future agricultural pest management practices [147].

3.2. Nanoparticles in Food Industry

NPs in combination with other technologies can bring impactful innovations in the
production, storage, packaging and transportation of food products. Food processing
transforms raw food ingredients into a palatable format with long shelf-life and in turn,
ensure efficient marketing and distribution systems for the enterprise. Fresh foods, on the
other hand, require robust logistics for their transportation from source to consumer. Nan-
otechnology based systems play an important role to maintain the functional properties by
incorporating NP based colloids, emulsions, and biopolymers solutions. Nanotechnology
has provided with a new dimension and ample opportunities to develop NPs for various
applications with deeper knowledge of the material. Application of nanotechnology in
food industry is based on nanostructures which target food ingredients as well as sensors.
Nano-food ingredients cover a wide area of applications starting from processing of food
to its packaging. Nanostructure based application in food processing comprises the use as
antimicrobial agents, nanoadditives, nanocarriers, anticaking agents, and nanocomposites
while in food packaging, they are applied as nano-sensors for monitoring the quality of
Catalysts 2021, 11, 902 13 of 35

food produced [182]. The nano materials can also serve as enzyme-supports because of
their large surface-to-volume ratio with respect to their conventional macro-sized counter-
parts. Recent nano-carriers have the potential to function as selective and exclusive delivery
systems in order to carry the food additives into the food ingredients without altering the
basic physicochemical properties and morphologies. For delivery of the bioactives to the
target sites, particle size is the most essential factor that affects the delivery rate as the
micro particles cannot be assimilated in some cell lines. A number of researchers have
developed various techniques of encapsulating these bioactives using nano-sized particles
or nano-emulsions resulting in enhancement of their bioavailability due to the increased
surface to volume ratio. Nanoencapsulation using nano-spray-drying is another promising
technique for the development of nanoparticles which can serve the food industry for the
production of bioactive ingredients.
However, challenges related to performance and toxicity of nanomaterials need to be
addressed to induce active development and applications of NPs. Additionally, legislation
for regulating the production, application, and disposal of nanomaterials for food industry
is of utmost importance. Public awareness and acceptance of the novel nano-enabled food
and agriculture products are also needed to be strengthened.

3.2.1. Application of NPs in Food Preservation and Packaging

The utility and global market of NPs have developed manifold in the recent years
and is expected to reach USD 125.7 billion by 2024. In the domain of food packaging, the
market is expected to reach a staggering USD 44.8 billion by 2030 [183]. Nanoceuticals and
Nutrition-by-nanotech are the available commercial names for food nano-supplements.
Nano-sized powders and nanocochleates are used for increasing absorption and delivery
of nutrients without altering the taste, flavor, and color of the food products. For better
absorption of micronutrients, vitamin spray-induced nanodroplets are used. The technique
of nano-encapsulation is involved when probiotics and similar targets are required to
deliver into the human system with the help of Fe and Zn nanostructured capsules. NP
based food supplements are more effective than their common counterparts because they
are able to react more efficiently with the human cells due to their nano-size.
Food preservation systems with antimicrobial packaging provide advanced barrier
properties to the food [184]. NPs or nanocomposite materials such as starch and sorbic acid-
based films are being utilized in various packaging applications for their microbial growth
inhibiting properties. They are effective due to their high surface-to-volume ratio as well
as enhanced surface reactivity of the nano-sized antimicrobial agents which assists in inac-
tivating microorganisms more efficaciously in comparison to micro- or macroscale agents.
Metallic and semiconducting NPs are the commonly used antimicrobial NPs. Metallic NPs
such as Ag with Cu, Au, and Pt demonstrate different degree of efficacies. Among the
semiconducting NPs, TiO2 , ZnO, WO3 , and MgO are proven antimicrobial agents. Other
antimicrobial NPs consist of natural biopolymers like chitosan (CTS) and enzymes (perox-
idase, lysozyme), organically modified nanoclay (e.g., quaternary ammonium-modified
MMT, Ag-zeolite), natural antimicrobial agents (e.g., nisin, thymol, carvacrol, isothio-
cyanate, and antibiotics) along with synthetic antimicrobial agents (quaternary ammonium
salts, ethylenediaminetetraacetic acid (EDTA), propionic, benzoic, and sorbic acids) [185].
Hybrid metal-polymer matrices is a new class of materials for diverse applications due
to their distinct properties such as high surface areas, orderly crystalline structures, and
pores with regular size and shape. Sensor composed of graphene oxide-nickel nanopar-
ticle biopolymer films is capable of measuring glucose concentration in the body fluids.
Nevertheless, it can also be employed in food application system because of the use of
biocompatible materials low toxicity of Ni and cost-effective technology [186,187]. The
antibiotic resistance mechanisms are irrelevant to the development of NPs since their mode
of action is only to stay in direct contact of the microbial cell walls without penetrating it.
The barriers that the natural NPs create can control microbial growth and consequently
spoilage of pathogens. Ag-NPs are used in biotexiles, electrical appliances, refrigerators,
Catalysts 2021, 11, 902 14 of 35

and other kitchen-wares as they act in bulk form and their ions have the ability to inhibit a
wide range of biological processes in bacteria [188]. The incorporation of AgNPs into the
gelatin-based nanocomposite film promisingly enhanced its antimicrobial activity. Further,
it was observed that nanocomposites, thus developed, showed potential antibacterial
properties against both Gram-negative and Gram-positive food-borne pathogens [189].
ZnO NPs have antibacterial nature which increases with decreasing particle size that can
further be stimulated using visible range light to incorporate in various polymers includ-
ing polypropylene [190]. The contamination of Escherichia coli can be inhibited by using
TiO2 as a coating in packaging material, and in combination with Ag, it improves various
disinfection processes.
Recent development of smart packaging, viz., oxygen scavengers, moisture absorbers,
and barrier-packaging products, account for 80% of the market share. Bakery and meat
industries significantly use nano-enabled packaging technologies. The food environment
is so enabled that it can continuously sense oxygen content, temperature, and micro-
bial load. Some examples include Ag-NP-incorporated enzymes for microbial detection
and gas sensing and nanofibrils of perylene-based fluorophores for detecting gaseous
amines from fish and meat spoilage. Additionally, ZnO and TiO2 nanocomposites are used
for detection of volatile organic compounds. Applications of NPs in the food industry
are relatively recent and have demonstrated rapid developments in this area [191]. The
major developments in this area include texture alteration, components and additives
encapsulation, enhancing sensory acceptance, controlled release of flavor, and enhancing
bioavailability of micronutrients [192]. NPs have also altered the novelty of packaging
materials enhancing their mechanical barrier and antimicrobial efficacy. Hence, the recent
advancements using NPs in food preservation and packaging may be used to overcome the
disadvantages of the biopolymer-based packaging technologies. Nanocomposites exhibit
enhanced barrier and mechanical and thermal properties compared to their polymers and
conventional counterparts.

3.2.2. Applications of NPs in Food Supplements and Value Addition

The applications of nanotechnology and the use of NPs in food science and technol-
ogy appear to have emerged from various sectors viz., pharmaceuticals, cosmetics, and
nutraceuticals. The advent of nanomaterials, which can interact with biological entities
at a near-molecular level makes it a common technology almost for various industries
including the above. Current nanotechnology applications in food industry for develop-
ing nanotextured food constituents as well as the delivery systems for nutrients require
techniques of nanoemulsions, surfactant micelles, emulsion bilayers, and reverse micelles.
The nanotextured food ingredients claim to offer better texture, taste, and overall accept-
ability [193]. Low fat nanotextured spreads, mayonnaise, ice creams, and similar products
claim to be as “creamy” as their full-fat alternatives, while offering a healthier alternative
to the consumers.
Nanocochleates (50 nm in size), known to protect micronutrients and antioxidants
from degradation during processing and storage, are based on a phosphatidylserine carrier
derived from soybean and are generally regarded as safe. The Greek term “cochleate”
means a ‘snail with a spiral shell’. It can be derived by adding calcium ions to small
phosphatidylserine vesicles in order to influence the formation of discs which are then
fused to large sheets of lipid molecules and finally rolled up into nanocrystals.
In another instance, self-assembled nanotubes were developed from a protein namely
lactalbumin which is a natural alternative for nanoencapsulation of pharmaceuticals,
nutrients, and supplements [194]. Nanotechnology comprises another major area, namely
nanoencapsulation, which is effectively used for delivering susceptible food ingredients
and additives. Microencapsulation can be employed to mask the taste and odor of tuna
fish oil for enabling it to be used for supplementation for its rich omega-3 fatty acid content.
Nanoencapsulated food ingredients and additives are used in a range of food products such
as the delivery of live probiotic microbes for healthy metabolic function. Nanoemulsion is
Catalysts 2021, 11, 902 15 of 35

another use in food technology to improve the quality of sweeteners, processed foods, and
beverages [195–197]. A summary on application of NPs in various aspects of food science
and technology is given in Table 3.

Table 3. Applications of NPs/Nanotechnologies in various aspects of Food Science and Technology.

Application NPs/Nanotechnology Function Reference

Antimicrobial, coating in packaging material,
TiO2 [198]
detection of volatile compounds
Quality enhancement of beverages,
Food Production Nanoemulsion [195–197]
sweeteners, and processed food
Enhancement of taste, color, and odor of food
Nanoencapsulation [194]
materials
Packaging of meat, fruit, and dairy products
AgNPs, Ag–ZnO NPs by AgNPs—doped nondegradable and edible [199]
polymers and oils; antimicrobial property
Low-density polyethylene film + Ag, ZnO
Orange juice, blueberry, strawberry [200–202]
NPs, TiO2 , kaolin
Chicken, pork, cheese, lettuce, apples, peels,
Ethylene vinyl alcohol + AgNPs [203]
eggshells
Polyvinylchloride + AgNPs Minced beef [204]
Food preservation and Fresh apples, white sliced bread, fresh carrots,
packaging Polyethylene + Ag, TiO2 NPs soft cheese, atmosphere packaging milk [205,206]
powder, fresh orange juice
Meats, cheese, confectionery, cereals,
boil-in-the-bag foods, extrusion-coating
Nanoclay-polymer nanocomposites [207]
applications for fruit juices and dairy products,
bottles for beer and carbonated drinks
Nanostorage containers, bakeware, containers,
Ag-ZnO NPs [199]
cutting boards
ZnNPs Preservation and transport [208]
Colloidal metal nanoparticles Enhanced uptake -
Nanopowders Increase absorption of nutrients -
Food supplement and value Cellulose nanocrystal composites Drug carrier -
addition Drug delivery, enhancement of taste and color
Nanocochleates -
of food materials

Various applications of NPs and nanotechnology provide numerous advantages for

food quality and safety. From farm to fork, nanotechnology is proven impactful at every
stage of food manufacturing, enhancing shelf-life, nutrition, quality control, and smart
packaging. However, the unregulated applications of NPs can pose potential risks to
human health and environment. Numerous studies have demonstrated the toxicological
effects of NPs on biological systems. Since food contact materials are already available
in the market in some countries, more data on the safety of such engineered NPs on
human health are necessary to implement regulations for such products. More research on
ecotoxicological effects of NPs will add on to the existing knowledge.

3.3. Drug and Medicine

Nanotechnology-based drugs have attracted a lot of attention in the last decade. The
unique properties of NPs, viz., small size, ability to travel through fine blood capillaries,
vessels, junctions, and barriers, have made them one of the most researched and studied
domains [209]. They have great advantages in terms of improvement of bioavailability
of drugs, solubility, toxicity safeguard, pharmacological activities, distribution, and pre-
vention from chemical and physical degradation and increased stability of drugs inside
the body [210]. Nanomedicines have shown higher capacity to bind with biomolecules
as well as reduction of inflammation/oxidative stress in tissues. Thousands of different
nanomedicines have been designed over the years; they have various applications in differ-
ent types of diseases. Few are approved for clinical use, and many more are in the phase of
clinical trials.
The use of nanomaterials as drugs and medicine implies nanotechnologies for medical
application with highly advanced medical intervention at molecular levels to cure diseases.
Catalysts 2021, 11, 902 16 of 35

It provides a platform for the discovery of therapeutic nanomaterials or nanomedicines.

The growth in nanomedicines has introduced numerous possibilities in medical sciences,
specifically in the drug delivery mechanisms. Their structural characteristics make them an
excellent mode for targeting at specific sites and quick penetration inside the cell/diseased
sites [211]. Depending on their application and origin, various types of NPs were discov-
ered based on therapeutic need. Liposomal, polymeric protein, metal based, and iron oxide
NPs have emerged as top-notcher. In this review, our discussion is mainly focused on the
application of metal NPs.

3.3.1. Silver NPs (AgNPs)

Silver NPs are considered ideal because of their unique properties like catalytic activity
and stability. They also contain anti-viral, anti-bacterial, and anti-fungal properties. One of
the applications of AgNPs is used in the antibacterial nanodevices, because of its Ag+ ion
effect. They can be positively used as anti-cancer agents due to their anti-proliferative effect
and ability to induce cell death [212]. AgNPs can be loaded/coated to reduce their toxicity
and improve their biological retention time which allows specific targeting of cancerous
cells. AgNPs from Andrographis echioides have been shown to inhibit the growth of MCF-2
cells and are widely used in human breast adenocarcinoma cell lines [213]. The viability of
tumor cells declines with increase in AgNPs concentration. Allium sativum AgNPs have
shown positive outcome in gastrointestinal carcinoma [214].

3.3.2. Gold NPs (AuNPs)

Gold NPs have anti-cancer properties and induce oxidative stress. They absorb
photons and convert those incident photons to heat that destroys cancerous cells. Cationic
gold NPs (2 nm diameter) are toxic at some dose [215]. Gold NPs exist in non-oxidized state.
Smaller NPs had less protein-to-protein ratio as compared to larger ones. Reports show that
gold NPs treated with Hela cervical carcinoma demonstrated increased reactive oxygen
species (ROS), leading to oxidation of lipid, proteins, and other several molecules [216].
AuNPs of 10 nm size were widely distributed in organs whereas 50–250 nm (large) NPs
were found to be distributed in liver, spleen, and blood when intravenously injected.

3.3.3. Iron Oxide NPs

Iron oxide NPs size lies between 1 and 100 nm in diameter. Their two main forms
are magnetite (Fe2 O3 ) and maghemite (γ-Fe2 O3 ). Iron oxide NPs were synthesized by
the process of precipitation in isobutanol (acting as surfactant) along with ammonium
hydroxide and sodium hydroxide [217]. Iron oxide NPs are unstable in aqueous media
without any surface coating; they aggregate and precipitate in vivo. The aggregates that
are formed by unstable iron NPs inside the blood are sequestered by macrophages. Iron
oxide NPs must be coated with different moieties to minimize the aggregation in certain
conditions. Iron oxide magnetic NPs have many applications in anti-cancer strategy
called hyperthermia; they destroy tissues nearby by generating heat. Iron oxide NPs have
many properties like high solubility, stability, distribution, biocompatibility, and prolonged
circulation time [218]. To increase the in vivo tumor imaging sensitivity, it is important to
deliver large concentration of NPs in both tumor cells and in tumor mass. In a study, it was
found that iron oxide NPs cause significant cellular morphological modifications, inducing
apoptosis and necrosis in MCF-7 cell lines [219].
However, there is a need for restraint on the use and applications of these nanomateri-
als as drugs. A detailed understanding on possible hazards and toxicological impact of
NPs on the environment as well as human health is needed prior to its application. Under-
standing the mechanisms of NPs access into the body, their function at cellular level, and
their influences on public health is the call of the hour. Nanomaterial’s characterization and
understanding their surface functionality inside living systems are critical to understand
their possible toxicological effects. All these parameters need more detailed studies before
the approval of nanomaterial-based drugs for human usage.
Catalysts 2021, 11, 902 17 of 35

3.4. Wastewater Treatment Process

The increase in population growth rate, industrialization, and excessive use of chemi-
cals has contaminated the aquatic environment by releasing wastewater to the environment.
The water from natural resources is not suitable for consumption due to the presence of or-
ganic (dyes, pesticides, surfactants, etc.), inorganic (fluoride, arsenic, copper, mercury, etc.),
biological (algae, bacteria, viruses, etc.), and radiological contaminants (cesium, plutonium,
uranium, etc.) [220,221]. Figure 6A depicts some of the common contaminants found in
water. Several techniques such as physical, chemical, and biological have been adopted for
the treatment of wastewater. However, search for new efficient technologies to improve
water purification at low-cost is the current research focus. Currently, nanotechnology
provides a new strategy for the removal of contaminants from wastewater with high effi-
ciency. Several approaches have been developed in combination with various NPs for the
successful removal of contaminants from wastewater as shown in Figure 6B.

Figure 6. Common pollutants found in water and the treatment processes. (A) Common pollutants present in water.
(B) Treatment approaches used for wastewater using nanoparticles.

The adsorption process is defined as the attachment of gaseous or liquid molecules

over the surface of the solid and forms a layer or film of molecules. This process is
mainly limited to the surface of the adsorbent where the adsorbate accumulates. The
adsorption process could be physisorption or chemisorption depending upon the nature
of bonding between the adsorbate and adsorbent i.e., van der Waals forces, covalent
bonding, or electrostatic attraction. Adsorption is the most commonly used technique for
the removal of contaminants from water due to its low-cost, easy operation, and absence
of secondary pollutants formation [146]. Due to the development of nanotechnology
and its wide applications in the past few decades, several nanostructured materials have
been explored as adsorbent for their potential application in the treatment of industrial
effluents, surface water, groundwater, and drinking water [222,223]. Nanoadsorbents
exhibit higher efficiency and faster adsorption rate compared to the conventional adsorbent
due to their small size, high porosity, and large active surface area [224]. Additionally,
these nanoadsorbents show high reactivity and catalytic efficiency. Nanomaterials such as
carbon nanotubes (CNTs), ferric oxide (Fe3 O4 ), graphene, titanium oxide (TiO2 ), manganese
oxide (MnO2 ), zinc oxide (ZnO), and magnesium oxide (MgO) are successfully used as
adsorbent for the removal of contaminants such as heavy metals, azo dyes, etc. from
the water [223,225]. Several nanosized metal oxide adsorbents including ferric oxide,
aluminum oxides, manganese oxides, titanium oxides, magnesium oxides, and cerium
oxides are proved to be promising for the removal of pollutants from water [226,227].
Furthermore, different metal oxide NPs are superparamagnetic which allows the easy
separation of these adsorbents from the reaction mixture with the application of an external
magnetic field. Das et al. [228] reported the removal of methylene blue dye, Cu(II), and
Catalysts 2021, 11, 902 18 of 35

Co(II) from aqueous solution using green synthesized magnetite NPs from crude latex
of Jatropha curcas and leaf extract of Cinnamomum tamala. The removal of Cd(II) from
contaminated solution was studied by using silver NPs prepared using leaf extract of Ficus
tree (Ficus Benjamina) [229]. In a similar type of study, the removal of cadmium ions from
contaminated solution was carried out using iron oxide NPs prepared by co-precipitation
method with tangerine peel extract [230]. Maximum removal efficiency 90% achieved
at pH 4.0 and adsorbent dose of 0.4 g/100 mL. Zinc oxide NPs synthesized from Aloe
vera and Cassava starch used as copper ion adsorbent and higher removal efficiency was
observed for Aloe vera synthesized NPs with the increase in adsorbate concentration [231].
These nanoadsorbents demonstrate remarkable efficiency in the removal of pollutants from
wastewater; however, the toxicity of residual NPs in the wastewater and reduced potential
activity due to the use of a huge number of NPs in the treatment process to minimize the
process duration are the major shortcomings of this process [232].
Filtration of contaminated water or wastewater through membranes is another way
to remove the pollutants from the water. Nanofiltration is efficient and effective for the
removal of different types of contaminants (organic, heavy metals, pathogens, etc.) from
wastewater, and the removal efficiency is mainly dependent upon the pore size and charge
characteristics of the membrane [233]. Numerous studies have focused on the development
and use of a composite membrane, prepared by the introduction of NPs into the polymeric
or inorganic membranes for the treatment of water. The incorporation of metal oxide
NPs like silica [234], alumina [235,236], zeolite [237], and TiO2 [161,238] into polymeric
membrane improved the membrane hydrophobicity and permeability. In addition to this,
the incorporation of antimicrobial NPs like silver NPs into membrane matrix hinders
bacterial attachment and biofilm formation [239,240].
Metal NPs are extensively used as nano-catalysts in water treatment due to their high
surface-to-volume ratio and surface catalytic activity. These nano-catalysts improve the
quality of water by degrading various contaminants, viz. dyes, pesticides, herbicides,
polychlorinated biphenyls, nitro aromatics, etc. [241]. Various kinds of nano-catalysts such
as electrocatalysts, photocatalysts, and Fenton-based catalysts are employed in the wastew-
ater treatment process [220]. The mechanism behind photocatalysis is the photoexcitation
of electron present in the catalysts. The light irradiation causes the generation of holes (h+ )
and exited electrons (e− ). Further, the generated holes (h+ ) are trapped by water molecules
(H2 O) in aqueous media that subsequently form the hydroxyl radicals (• OH) [242]. These
hydroxyl radicals are highly reactive and powerful oxidizing agents which oxidize the or-
ganic pollutants leading to the formation of water and gaseous degradation products [242].
Numerous studies reported photocatalytic activity of green synthesized Ag, Au, Pt, and Pd
NPs in degradation of different dyes [63,243–247]. Additionally, various metal oxide NPs
such as ZnO, CuO, FeO, SnO2 , TiO2 , NiO, CeO2 , etc. exhibited excellent photocatalytic
activity for the degradation of different organic pollutants [248].

3.5. Antimicrobial Activity

In the past decade, application of nanomaterials to control microbial proliferation has
garnered much interest from scientists worldwide [249,250]. The increase in resistance of
microorganisms to antimicrobial agents, including antibiotics, has led to a spike in health-
related complications. A vast body of work has revealed that by combining three forces
of material science, nanotechnology, and the inherent antimicrobial activity possessed by
certain metals, innovative applications for metal NPs can be identified [251]. Previous
studies have reported that metal and their counterpart metal oxide nanoparticles have
displayed toxicity towards numerous microorganisms [209,250]. These NPs may be used
successfully to stop the growth of various bacterial species.
The surge in development of multi-drug resistant pathogens is presenting itself as a
grave problem to public health, and thus, several studies have been conducted at improving
the prevailing antimicrobial treatments [251]. It has been identified that approximately 70%
of bacterial infections have developed resistance to one or more of the first- and second-line
Catalysts 2021, 11, 902 19 of 35

drugs that have been traditionally used to treat the infection [252]. The development of
resistance in bacteria to commonly used chemical antibacterial agents may occur due to
the lengthy production-consumption cycle, thus leading to reduction in efficacy. Moreover,
the rampant use of poor quality or over-the-counter medicines in developing countries has
led to a steep rise in antimicrobial resistance [253]. The need of the hour is to speed up
the research and development and the synthesis of novel antimicrobial agents which are
effective as well. NPs as antibacterial agents have turned out to be an emerging technology
against this challenge, which have the ability to establish an effective nanostructure, which
may be used to deliver the antibacterial agents, hence targeting the bacterial growth locally
and more efficiently. In addition, nanoparticles have proved to have the potency that it
leaves the pathogens with little device to develop resistance against them. Most of the
available metal oxide NPs have zero toxicity for mammalian cells at the concentrations
that have been used to kill bacterial cells, which in turn is an advantage for using them at a
larger scale [254].
Metals like gold (Au), silver (Ag), titanium (Ti), copper (Cu), and zinc (Zn) are known
to have their own properties and potency and display differential activity against microor-
ganisms. This information has been understood and utilized across various cultures for
centuries [255]. Numerous kinds of nanoparticles and their derivatives have been explored
for their potential antimicrobial effects against several microorganisms. Metal nanoparticles
such as gold (Au), silver (Ag), silicon (Si), silver oxide (Ag2 O), titanium dioxide (TiO2 ), zinc
oxide (ZnO), copper oxide (CuO), calcium oxide (CaO), and magnesium oxide (MgO) have
been recognized to display antimicrobial activity. In vitro studies have suggested that metal
nanoparticles have the potential to inhibit several microbial species, like Escherichia coli,
Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, etc. [256–265].
The type of materials used in formulating the nanoparticles along with their particle
size are the two most significant parameters, which can have an effect on the effectiveness
of antimicrobial activity. It is well established that nanoparticles tend to possess different
characteristics when compared to the same material having significantly greater dimen-
sions. This is because the surface to volume ratio of the NPs considerably increases with
a decrease in the particle size [266]. Certainly, in dimensions of nanoscale, the fraction
of the molecule surface noticeably increases, which in turn can lead in improvement of
some of the properties of the particles. For example, it may be mass transfer, heat treat-
ment, catalytic activity, or the dissolution rate [267]. Additionally, the morphology and
physicochemical properties of NPs have also been demonstrated to wield an effect on their
level of germicidal activities. Literature survey has pointed that the particle size plays
a role as vital parameter that can determine the effectiveness of antimicrobial activity of
the metal nanoparticles [268,269]. The use of combination therapy with metal nanoparti-
cles has the potential to be a strategy that can help tide over the emergence of bacterial
resistance to multiple antibacterial agents [270,271]. More studies need to be developed
to understand if green synthesized nanoparticles have better efficacy over traditionally
synthesized nanoparticles. Current studies have displayed the same level of antimicrobial
effects [272,273].
The shape of nanoparticles also has major influence on their antimicrobial effects [268].
Several research studies have investigated these shape-dependent characteristics of nanopar-
ticles. A study described antibacterial activity of Ag NPs in three dissimilar shapes, namely,
spherical, rod-shaped, and truncated triangular. It came to the conclusion that the truncated
triangular NPs were more inclined to be reactive owing to their high atom density surfaces
and consequently displayed greater antimicrobial activity [274]. In another study, the
size and shape-dependent antimicrobial activity of fluorescent Ag nanoparticles (1–5 nm)
was studied against some selected Gram-positive and Gram-negative bacteria [275]. They
highlighted that the size and shape of the particles generated an effect on its activity. These
investigations reported that the smaller the particles size, the easier they breach the cell
wall exhibiting heightened antimicrobial activity. Furthermore, the authors proposed that
these AgNPs could be used for multiple diverse procedures such as wound dressing,
Catalysts 2021, 11, 902 20 of 35

biofilms, bio-adhesives, and coating of certain biomedical materials. It was also found
that antimicrobial property of TiO2 is related to the size, shape, and structure of its crys-
tal [252]. It is proposed in this particular study that the generation of ROS, ultimately
leading to development of oxidative stress in the cells, may be a significant mechanism for
TiO2 nanoparticles to show its germicidal activity. It is well known then that ROS has the
capability to cause site specific DNA damage, ultimately leading to the death of the cell.
The exact mechanisms in which nanometals present the antibacterial effect are still an
area of active investigation. However, two common options have been proposed in this
aspect. Firstly, toxicity associated with free metal ions can arise due to the dissolution of
the metals from the surface of NPs. Secondly, oxidative stress could be triggered through
the production of reactive oxygen species (ROS) on the surface of the NPs. Based on
literature review, there are some intrinsic factors that can have an influence on the ability
of nanomaterials in reducing the number of cells or completely eliminating the cells [255].
NPs are a promising technology, and owing to its vast application, understanding
nanotoxicity and its consequences is of utmost importance. For decades, the pharmaceu-
tical industry has used NPs as a tool to reduce toxicity and side effects of drugs [276];
nonetheless, one needs to be careful when using NPs, as certain safety concerns still exist.
Several reports have identified damage to neurological and respiratory organs issues in
the circulatory system. In addition, other yet unknown toxic effects of NPs are few of the
foremost apprehensions in using NPs as part of a systemic therapy. Undeniably, numerous
NPs seem non-toxic, and a few of them are reduced to having non-toxic properties, which
ultimately has beneficial effects on health [209,277–279]. Nevertheless, further studies need
to be executed focusing on minimizing the toxicity of metal and metal oxide NPs, that
will eventually be applied in therapy as a proper substitute to disinfectants and antibi-
otics especially in biomedical applications. Moreover, current research should make the
application of antimicrobial activity of NPs in eradication of microbial infections as one of
their priorities.

4. Toxicity of Metal Nanoparticles

NPs have wide applicability in different sectors such as electronics, agriculture, chem-
icals, pharmaceutical, food, etc. due to their unique physicochemical properties [280].
The most commonly used NPs by various sectors include metal oxide NPs such as
silicon oxide (SiO2 ), titanium dioxide (TiO2 ), zinc oxide (ZnO), aluminum hydroxide
[Al(OH)3 ], cerium oxide (CeO), copper oxide (CuO), silver (Ag), nanoclays, carbon nan-
otubes, nanocellulose, etc. [281,282]. However, massive release of NPs into the environment
(air, water, and soil) by various industries is resulting in production of nanowaste and
proving to be dangerous for the living organisms and causing threat to ecosystem balance.
Various characteristics of NPs affecting their toxicity are size, nature, reactivity, mobility,
stability, surface chemistry, aggregation, storage time, etc. NPs cause adverse consequences
on human health and animals. Use of NPs has intensified the risk of various diseases in
humans such as diabetes, cancer, bronchial asthma, allergies, inflammation, etc. [3]. The
animal reproductive system has also been shown to be affected due to toxicity of various
NPs such as Au, TiO2 , etc. [283,284]. NPs enter the animal body through ingestion and
inhalation and get absorbed by the cells through the processes of phagocytosis and endocy-
tosis and induce the generation of reactive oxygen species (ROS), ultimately resulting into
lipid peroxidation, mitochondrial damage, etc. Different NPs such as Ag, Cu, ZnO, Ni, etc.
have also reduced the enzymatic activity of various microorganisms. In addition, excessive
production of NPs is also affecting the food web of the ecosystem [285]. Toxicity effects of
NPs over plants, animals, and microorganisms are shown in Figure 7.
Catalysts 2021, 11, 902 21 of 35

Figure 7. Toxicity effects of NPs on microbes, plants, and animals.

4.1. Impact of Nanoparticle Toxicity on Plants

Plants are of high fundamental significance as they perform photosynthesis and
release oxygen in the atmosphere. As all the plant parts (roots, shoots, and leaves) are
in direct contact with environmental matrices (air, water, and soil), they may get affected
more by the NPs contamination as compared to the other living beings. NPs present in the
atmosphere can enter into the plant body through the stomatal openings on leaves [286],
while those present in soil and water can be selectively up taken by the plant roots [287].
NPs proved to be toxic to plants and hamper their growth and development. The
toxicity in plants is mainly due to ROS production, causing lipid peroxidation and ulti-
mately leading to DNA damage, reduction in photosynthetic pigments, plant biomass,
soluble protein content reduction, etc. [288]. However, plants have a defensive system
against oxidative stress in the form of enzymatic and non-enzymatic antioxidants, which
may become inefficient under higher oxygen concentrations [289].

Assessment of NPs Phytotoxic and Genotoxic Effects on the Plants

While phytotoxic effects of NPs on the plant can be assessed by analyzing morpholog-
ical and physiological changes, genotoxicity can be observed by observing DNA damage
in the plant cell. By evaluating various physiological parameters such as germination,
biomass production, leaf number, photosynthetic ability, root and shoot length, etc., phy-
totoxicity levels of NPs on the plants can be assessed. To observe genotoxic effects on
the plant, assessment of cytology of plant roots for the determination of mitotic index,
chromosomal abnormalities, etc. is the simplest approach [290].
SiO2 NPs are among the top metal oxide NPs produced by various industries such
as cosmetics, pharmaceuticals, food, etc. due to their worldwide demand. Positive effects
of SiO2 NPs on various plants such as Oryza sativa, V. faba, S. lycopersicum, Medicago sativa,
etc. have been shown in different studies [291–294]. Toxic effects of SiO2 NPs on plants
have been investigated by Slomberg and Schoenfisch [295] in Arabidopsis thaliana, Karimi
and Mohsenzadeh [296] in Triticum aestivum, and Silva and Monteiro [297] in A. cepa, etc.
Regarding TiO2 NPs, negative effects have been shown in Z. mays and Vicia narbonensis [298],
V. narbonensis [299], V. faba [300,301], etc. ZnO NPs are majorly used in agriculture sector
(as pesticides, fertilizers, etc.) [302].
In green algae, Chlorella vulgaris and Dunaliella tertiolecta, AgNPs proved to be in-
hibitory for their growth and displayed higher ROS production and lipid peroxidation [303].
AgNPs also proved to be toxic for the growth and development of green algae,
Pithophora oedogonia and Chara vulgaris [304].

4.2. Toxicity of Nanoparticle-Based Drugs

The commercial applications of NPs as therapeutics for treatment of diseases is a
double-edged sword. Even though many studies are being done worldwide to analyze the
toxic effects of NM exposure, the possible mechanism of NMs interactions with biological
systems and their consequences are still unknown. Research has shown that NPs can travel
Catalysts 2021, 11, 902 22 of 35

through the bloodstream and easily cross membrane barriers. This in turn can adversely
affect tissues and organs at molecular and cellular levels [305]. NPs have demonstrated
the capacity to cross the blood–brain barrier (BBB) and gain access to the brain [306].
Small size, large surface area to mass ratio (SA/MR), and surface characteristics determine
nanoparticle’s interaction with biological milieu and the resultant toxic effects that ensue.
The unique nature of the NMs allow them to easily pass-through cell and tissue membranes
and cellular compartments to cause cellular damage. The large SA/MR of NPs also remains
open for active chemical interactions with cellular macromolecules. Increase in surface
area of the identical chemical further enhances adsorption properties, surface reactivity,
and potential toxicity [305].
NPs have the tendency to translocate across cell barriers from the entry point i.e., the
respiratory tract to secondary organs, reach the cells by various mechanisms and start
interacting with subcellular structures. These properties make NPs uniquely suitable for
therapeutic and diagnostic uses. NPs are transported neuronally, involving retrograde
and anterograde movement in axons and dendrites as well as perineural translocations.
The target organs such as the central nervous system (CNS), however, bear the brunt of
potential adverse effects (e.g., oxidative stress) [306]. The size of NPs has an important
role in renal clearance and in avoiding immune activation, enhancing the efficacy and
circulation time of the drug inside systemic circulation [209]. In case of nickel NPs, small
particle sizes (less than 200 nm) are preferable for entering into epithelial cells, whereas
larger NPs are phagocytosed by macrophages present. Another issue is surface charge
which restraints the fate of NPs. Positively charged surface NPs with amorphous nature
do not enter inside the cells, whereas, negatively charged crystalline nickel sulphide and
sub-sulphide particles can enter cells by phagocytosis. Inhalation of MnO2 NPs leads to
the formation of ROS causing oxidative stress in brain [210].
Silver NPs of different sizes, i.e., 20 or 40 nm (Ag20Pep and Ag40Pep) were analyzed in
THP-1-derived human macrophages through their cellular uptake. Results demonstrated
a majority of the AgNPs spread throughout the cells. Formation of protein carbonyls or
induction of heme oxygenase I are some of the associated responses due to oxidative stress
are also observed. The charged Au NPs sized 15 nm cause cell death by apoptosis, whereas
neutral Au NPs cause necrosis in HaCaT (human epidermal keratinocyte) cell lines [307].
Several NPs can penetrate inside the nuclear envelop, and they play an important role
in inducing genotoxicity. Silver NPs have been found to be more toxic than gold NPs.
TiO2 NPs are considered as biological inert material in vitro and in vivo, while TiO2 NPs
larger than 15 µm are highly toxic, generating ROS. TiO2 is toxic to PC12 cells [308]. It was
reported that toxicity of NPs increases with increasing surface charges, i.e., lower positive
charge NPs have less electrostatic interaction with the cells. Positive ZnO NPs have more
cytotoxic effect in A549 cells as compared to same sized and shaped negatively charged
ZnO [309]. Rod shaped Fe2 O3 NPs produce high cytotoxic responses compared to spherical
Fe2 O3 NPs in RAW 264.7 (murine macrophage) cell lines [310]. Rod-shaped CeO2 NPs cause
more toxic effects and produce LDH and necrosis factor-α in RAW 264.7 cells [209]. NPs
cytotoxicity depends on assay, cell line, and physical and chemical properties. Copper oxide
NPs have been found to produce toxic side effects in liver and kidneys when examined
on lab animals; after oral administration and interaction with gastric juice, they form
reactive ionic copper. It was reported that silver NPs and iron oxide NPs can penetrate
and cross the blood–brain barrier [306]. Iron oxide has the capacity to accumulate inside
liver, spleen, lungs, and brain and has the capacity to cross the BBB after inhalation [305].
Iron oxide shows less cytotoxic effects at high concentration (300–500 kg/mL, 6 h) than
in low concentration (25–200 µg/mL). At low concentration, it generates Reactive oxygen
species (ROS), DNA damage, and causes lipid peroxidation. Silica based NPs of size 70 nm
at 30 mg/kg concentration have been found to alter biochemical parameters [210]. Hence,
a number of studies show evidence of NPs causing DNA and membrane damage, protein
misfolding, and mitochondrial damage.
Catalysts 2021, 11, 902 23 of 35

In a clinical scenario, several factors need to be addressed, viz., bioavailability, adverse

reactions, cellular interactions, biodistribution, biodegradation, etc. The successful clinical
translation of nanomedicines is therefore a long and onerous process of weighing the
benefits against risks of toxicity involved. Scientists must proceed with caution and refrain
from premature launch of nanomedicines without assessing the adverse effects involved.
Compared to physio-chemical methods, biologically synthesized NPs have been
proved to be non-toxic/less toxic due to non-use of external stabilizing agents and haz-
ardous chemicals/solvents during the synthesis process. Hasan et al. [164] compared the
morphological, physiological, and biochemical responses of biologically and chemically
synthesized iron oxide NPs in Zea mays. The biological synthesized FeO NPs promoted
better plant growth as compared to the chemically derived NPs. The chemically synthe-
sized NPs proved toxic and hampered the plant growth even at lower concentrations. In
a similar kind of study, Anna et al. [311] observed better growth of green algae P. kessleri
while using biologically synthesized NPs compared to chemical synthesized NPs.

5. Major Challenges and Future Perspective

In the recent past, research on NPs and their potential applications have progressed
by leaps and bounds. Numerous studies have reported the green synthesis of metallic NPs
using various biological sources such as plants, bacteria, fungi, and yeast. However, several
challenges persist, which limit its large-scale production and consequent applications. Some
of the major challenges observed during the synthesis are summarized below:
• Detailed optimization studies on reactants (plant extract, microorganism inoculum,
fermentation medium composition, etc.) and process parameters (temperature, pH,
rotational speed, etc.) are required to control the size and shape of the NPs.
• Studies also need to be focused on enhancing various physicochemical characteristics
of NPs for specific applications.
• The involvement of each metabolite of plant extract and cellular components of
microorganism in the synthesis of NPs should be completely analyzed.
• Scale-up of NPs production for commercial purposes using green synthesis methods
needs to be prioritized.
• Improvement of NPs yield and stability with reduced reaction time is needed by
optimizing various reaction parameters.
Addressing these challenges could make the green synthesis methods cost-effective
and comparable to the conventional methods for the large-scale production of NPs. Ad-
ditionally, the separation and purification of NPs from the reaction mixture is another
important aspect that need to be explored. A detailed toxicological study of the NPs on
plants and animals is necessary for expanding its application in diverse fields. In addition to
wild type strains, genetically modified microorganisms with the ability to produce greater
quantity of enzymes, proteins, and biomolecules could further enhance the biosynthesis as
well as the stabilization of NPs. Further, enhancement of metal accumulation capacity and
tolerance of genetically modified microorganisms could provide a futuristic approach for
the production and application of metal NPs using the green synthesis method.

6. Conclusions
The present review focuses on the green synthesis of metal NPs derived from plants
and microorganism and their applications. Green synthesis methods provide a clean,
non-toxic, and eco-friendly approach for the synthesis of metal NPs compared to other
conventional techniques like physical and chemical methods. A wide range of plant
materials including leave extract, fruit extract, seed, fruit, bark, etc. and microorganism
such as bacteria, fungi, actinomycetes, etc. have shown potential for synthesis of various
metal and metal oxide NPs (viz., Au, Ag, Pt, Pd, Ni, Se, Cu, CuO, and TiO2 ). The size and
shape of NPs and the reaction rate strongly depend on various experimental parameters
such as reaction time, reactant concentration, pH, temperature, aeration, salt concentration,
etc. Different characterization techniques such as UV-VIS spectroscopy, FTIR, XRD, SEM,
Catalysts 2021, 11, 902 24 of 35

TEM, EDX, and AFM have been used to determine the shape, size, and morphology of
biosynthesized NPs. However, in terms of translational research, several factors, viz.,
bioavailability, adverse reactions, cellular interactions, biodistribution, and biodegradation,
need to be addressed. The accumulation of these NPs in the environment and their
uptake by biological systems can lead to disastrous consequences as a number of studies
show evidence of NPs causing DNA and membrane damage, protein misfolding, and
mitochondrial damage. Although numerous studies reported the biological synthesis of
metal NPs, a thorough investigation is the need of the hour for widening their applications
and successful commercialization.

Author Contributions: Conceptualization, P.K.D.; writing—original draft preparation, P.K.D., J.K.,

A.K.D., S.S. (Soumi Sadhu), S.S. (Sunita Sharma), S.S. (Swati Singh); writing—review and editing,
P.K.D., J.K., A.K.D., S.S. (Soumi Sadhu), S.S. (Sunita Sharma), S.S. (Swati Singh), P.K.G., B.S.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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