Iron Oxide Nanoparticles-Plant Insignia Synthesis with Favorable Biomedical Activities and Less Toxicity, in the “Era of the-Green”: A Systematic Review

In the era of favoring environment-friendly approaches for pharmaceutical synthesis, “green synthesis” is expanding. Green-based nanomedicine (NM), being less toxic and if having biomedical acceptable activities, thence, the chemical methods of synthesis are to be replaced by plants for reductive synthesis. Iron oxide nanoparticles (IONPs) exhibited remarkable anti-microbial and anti-cancer properties, besides being a drug delivery tool. However, owing to limitations related to the chemical synthetic method, plant-mediated green synthesis has been recognized as a promising alternative synthetic method. This systematic review (SR) is addressing plant-based IONPs green synthesis, characteristics, and toxicity studies as well as their potential biomedical applications. Furthermore, the plant-based green-synthesized IONPs in comparison to nanoparticles (NPs) synthesized via other conventional methods, characteristics, and efficacy or toxicity profiles would be mentioned (if available). Search strategy design utilized electronic databases including Science Direct, PubMed, and Google Scholar search. Selection criteria included recent clinical studies, available in the English language, published till PROSPERO registration. After screening articles obtained by first electronic database search, by title, abstract and applying the PICO criteria, the search results yielded a total of 453 articles. After further full text filtrations only 48 articles were included. In conclusion, the current SR emphasizes the perspective of the IONPs plant-mediated green synthesis advantage(s) when utilized in the biomedical pharmaceutical field, with less toxicity.


Introduction
Recently, IONPs have provided various applications in the medical field [1]. Owing to the limitations of IONPs conventional synthetic methods, namely, methods of synthesis and toxicity, there has been a great emphasis on finding alternative methods for IONPs' preparation. Literature has already reported on NPs' drawbacks processed with either the attrition or pyrolysis conventional methods [2]. These drawbacks include the formation of defective surface structures, use of toxic chemicals, upon processing, damage to the environment and humans, low-rate production, and high cost. Plant-mediated IONPs synthesis emerged as a suitable alternative synthetic method, during the last decade.
Review aim and objectives. In an attempt to highlight the possible benefits of utilizing plant-mediated IONPs green synthesis, the review will compare NPs characteristics, efficacy, Searched electronic databases were PubMed, ScienceDirect and Google Scholar. All records retrieved from e-database searches were downloaded locally and managed using the Mendeley X86 software facilities.
SR Registration. PROSPERO 2020 [CRD42020203760], obtained 11 September 2020. Inclusion criteria. Studies that utilized plant-based synthesis for preparation of IONPs and studies that investigated the efficacy or toxicity of plant-based synthesis IONPs or investigated their clinical activity. Exclusion criteria. Studies that utilized only chemical processing for preparation of IONPs and other biogenic methods, not plant-based, were excluded.

Effect Plant Family/Part Used NPs Size/Morphology Mechanism of Anti-Cancer Effects
Ref.

Biomedical Antioxidant
Corn of Z. mays L.
The biomedical effects of plant-based green-synthesized IONPs against some cancer hallmarks are addressed in Table 4. Figure 4 summarizes some cancer hallmarks acted upon by plant-based green-synthesized IONPs, attributed to the activity of the IONPs as well as the biological plant compound(s). Table 5 lists articles tested plant-based green-synthesized IONPs in vivo and in vitro toxicities.
Six studies appeared in the SR current search obeying the inclusion criteria to be included in the review, where IONPs were plant-based green-synthesized, but without reported biomedical applications, as listed in Table 6.

Discussion
The usage of plant extracts, as a green method, for IONPs synthesis is environmentally favored as well as being a stable economic method for NPs synthesis [3]. To fully exploit the potential of green-NPs synthesis, a detailed understanding of the principles of green IONPs synthesis and high-throughput screening of stabilizing/capping agents on the physicochemical properties is required.
The superiority of plant extracts for NPs synthesis (Figure 2) is depicted in Figure 3. Plant extract advantage(s) are (1) to act as a capping or stabilizing agent, thus reducing the NPs size and improving their reactivity [4,5]. Moreover, (2) plant extracts contain bioactive constituents ranging from essential oils, flavonoids, polysaccharides, alkaloids, terpenoids, minerals, amide glycosides, amino acids, fatty acids, sitosterol, tannins, glycosides, quinones, coumarins, carotenoid compounds, phenolic acid, lignans, saponins, and other polyphenols (Table 1), which is a coat surface to the NPs core, preventing agglomeration, and thus, help obtain more uniform particle size distribution [2,4,6], keeping in mind the beneficial medically approved and recommended effects of these bio-active nutraceuticals, by themselves, potentiating the formed IONPs action, in comparison to chemical-based synthesis.
Plant extract preparation, as illustrated in Figure 2, is executed after washing the plant part with tap water, then deionized water, to remove any dust or particulate matter. Second, after drying the plant parts well, they are chopped into small pieces [13], via mortar and pestle grinding [14][15][16]20] or homogenized by an electric grinder [7-9,24]. Finally, the obtained powder or paste is heated in sterile water for some time, till extraction is complete. The powder or paste is filtered through Whatman no. 1 filter paper, three times, and the obtained filtrate is chilled at 4 • C for further use.
Using plant extract to synthesize IONPs, through iron reduction and stabilization as NPs, by components of the plant extract, would give rise to either elliptical or spherical shapes, hexagonal [27], or cubical, rhomboidal shapes [28]. They could also exhibit a dendrimer shape, with branched surfaces at nanometer magnifications [29]; a structure may have resulted from chemical interactions such as hydrogen and electrostatic bonds between the organic capping agents of plant secondary metabolites and the IONPs core, a feature not present when IONPs were synthesized using the conventional methods.
The degree of plant-based green-synthesized IONPs aggregation is attributed to the super magnetic properties of iron, present with IONPs, competition between repulsive (electrostatic), and attractive (dipolar and Van der Waals) interactions among particles [7].
It is noteworthy mentioning that the IONPs plant-based green-synthesis relies on the co-precipitation method, in which iron ions, from iron chloride FeCl3, for example, are reduced in the presence of plant extracts. The plant extract might play the role of the reductant as well as the capping agent, due to the reducing properties of the bioactive constituents in the plant, minerals, and antioxidant properties as well.
However, the co-precipitation method has the physicochemical characteristics limitations, namely, poor crystallinity, low control on size, and polydispersity. However, the major advantage of using plant extract different moieties is in providing more biocompatible NPs to control the NPs diverse shapes.
The scheme in Figure 2 presents the mechanism of IONPs plant-based green synthesis. Where, a 2:3 volume ratio of 0.1 M FeCl 3 solution is to be added to the leaf extract, and by adding 1.0 M NaOH to the solution, the pH changed from 3 to 6. The formation of IONPs indicated by the appearance of a black color precipitate, separated by centrifuging the solution at 7000 rpm for 15 min. The obtained black precipitate is washed and freeze-dried at −40 • C at 10 Pa pressure for 24 h. The as-obtained IONPs stored in an airtight dry container for further characterization and use. Table 2 addresses plant-based green-synthesized IONPs biomedical efficacy studies with antimicrobial activity; anti-bacterial activity, where most of the current SR selected studies evaluated the anti-bacterial effect of green-synthesized IONPs, against Grampositive and/or Gram-negative bacterial strains. Veeramanikandan et al. (2017) investigated the effect of plant-based green-synthesized IONPs using Leucas aspera leaf extract [16], where these IONPs strongly inhibited the Gram-positive bacteria; Bacillus cereus, Staphylococcus aureus and Listeria monocytogens growth. On the other hand, IONPs moderately inhibited the growth of Gram-negative bacteria; Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, Shigella flexneri, Vibrio cholera and Pseudomonas aeuroginosa, at a concentration of 150 µg. However, IONPs showed a low inhibitory effect on the growth of Bacillus cereus. The antibacterial activity of IONPs synthesized using the aqueous fruit extracts of Hyphaene thebaica, is in the following order of B. subtilis > P. aeruginosa > K. pneumonia > E. coli > S. epidermidis, with an antimicrobial potential greater than the positive control (Erythromycin) [22].
The plant-based green-synthesized IONPs from the Glycosmis Mauritania leaf extract showed good anti-bacterial activity against Bacillus cereus, B. subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Proteus mirabilis, P. vulgaris, Pseudomonas fluorescence, Staphylococcus aureus and Vibrio fluvialis [8]. Being a popular source for green IONPs synthesis, the leaf aqueous extract of Artemisia haussknechtii Boiss showed a better effect against E. coli than S. aureus and S. marcescens [29]. The Silda cordifolia leaf extract mediated IONPs synthesis, and held potent antibacterial activity against various Gram-positive and Gram-negative bacteria; E. coli, K. pneumoniae, B. subtilis, and S. aureus, with comparable results to that of neomycin [12].
Couroupita guianensis Aubl. fruit extract synthesized IONPs exhibited a greater antibacterial effect against Gram-negative E. coli, S. typhi and K. penumoniae than Gram-positive S. aureus [24]. Ulva prolifera-derived IONPs showed a better effect than chemo-IONPs against bacterial strains in the following order: Staphylococcus epidermidis, followed by Bacillus subtilis, and Bacillus pumulis [30]. Green-synthesized IONRs showed the maximum antibacterial activity for S. aureus, while the minimum for P. aeruginosa [23].
Biogenic hematite NPs of average size <10 nm was synthesized using a green approach with Aloe vera extract (ALE). ALE-biogenic hematite NPs had prominent growth inhibition that is attributed to the smaller size, thinner protective layer surrounding the cells, higher dispersibility, and stability, owing to capping with organic moieties [28].
It is noteworthy to mention that the difference observed in the antibacterial potential of plant-based green-synthesized IONPs, against Gram-negative and Gram-positive bacteria, could be attributed to the differences in cell wall structure inherent in Gram-negative or Gram-positive bacteria. Where, Gram-positive bacterial cell wall possesses thick peptidoglycan layer, that contains teichoic and lipoteichoic acids, whereas, Gram-negative bacteria have thin peptidoglycan layer and the outer membrane contains lipopolysaccharides, phospholipids, and phosphoproteins.
Therefore, first, cannot conclude from the reported data if these NPs are more efficient against Gram-positive or against Gram-negative bacteria. Second, the antibacterial activity related to the plant-based synthesis cannot be ruled out. Third, the strength or specificity of the effect depends on the sensitivity of the microorganism. However, NPs, in particular oxide ones, show a tiny particle size and a large surface area, which can improve their antibacterial activity and surface reactivity, being a good choice nowadays following the prevalence of resistance to antibiotics. Therefore, a proposed recommendation for engineering IONPs with toxic surface modifiers, of plant origin or not, to improve their overall antimicrobial activity, is raised.
Plant-based green-synthesized IONPs with anti-fungal activity were reported in six articles. They checked the inhibitory effect of plant-based green-synthesized IONPs on fungi growth, as illustrated in Table 3, where these six studies mentioned the effect of green-synthesized IONPs on 11 different fungi. Plant-based green-synthesized IONPs exhibited anti-fungal activity via pronounced penetration ability through the fungal cell surface, followed by dissociation into respective ions, with generation of oxidative stress (OS) via production of reactive oxygen species (ROS), based on iron oxides within IONPs. Superoxide anions, hydroxyl radicals, and hydrogen peroxide (H 2 O 2 ) are ROS, that can damage biological components such as DNA, proteins, and lipids. Moreover, iron oxides disrupt enzymatic reactions within the bacteria.
This was the case when using green-synthesized IONPs utilizing Platanus orientalis [39] against Mucor piriformis and Aspergillus niger.
It is worth mentioning that the current anti-fungal activity is based on the production of ROS and the overall generated OS, which is the opposite case, when using greensynthesized IONPs for cancer treatment.
IONPs that were green synthesized using Laurus Nobilis leaves reported inhibition zones of 13 and 14 mm against Aspergillus Flavus and Penicillium spinulosumas, respectively. Meanwhile, nystatin reported a 20-mm and 19-mm inhibition zones against them, respectively [27].
If better anti-fungal activity was observed, when using chemically synthesized IONPs in comparison to the green synthesized ones, this would be explained on the basis of smaller particle size.
As a perspective, it is worth mentioning that biologically synthesized IONPs with standard antibiotics (kanamycin and rifampicin) can exert synergistic effects against the five foodborne pathogenic bacteria, enabling a reduction in the dose of antibiotics, leading to decreased bacterial resistance and the overall mammalian cell toxicity [13]. Moreover, IONPs could be used in combination with conventional anti-fungal agents in the clinical setting, since the amount of both agents can be substantially reduced and thus, potentially avoid the adverse effects caused by the use of high doses of conventional anti-fungal drugs, with better efficiency. This is together with the retained plant metabolites activities as phenolic compounds generating more ROS to exert antimicrobial effect by themselves. Moreover, flavonoids hydroxyls are important for metal-binding activity.
Plant-based green-synthesized IONPs target some hallmarks of cancer, as enumerated in Table 3 and illustrated in Figure 4. IONPs synthesized using the corn (Zea Mays L.) displayed a dose-dependent anti-proteasome potential (large multi-catalytic proteinase complex located in the cytoplasm) [13]. Nuclear transcription factor kappa-B-cell (NFk B) plays various roles in cellular homeostasis regulation. IONPs conventionally synthesized showed 50.3% NFk B inhibition, while IONPs synthesized from Rhus plant extract showed 57.5% NFk B inhibition [32]. Green-synthesized IONPs using the aqueous fruit extracts of Hyphaene thebaica were able to stop the activity of protein kinase enzyme [22]. The antioxidant activity (OS is one of the cancer hallmarks to be targeted) can be determined in vitro by measuring several free radical scavenging methods, such as 2,2 -azino-bis(3ethyl benzo-thiazoline-6-sulphonic acid (ABTS), nitric oxide, 1,1-diphenyl-2-picrylhydrazyl (DPPH), as well as the reducing power assays [20,40]. Green biosynthesized magnetic IONPs using the corn (Zea Mays L.) ear leaves aqueous extract showed lower ABTS scavenging potential compared to standard [13]. When fenugreek seeds extract was used to prepare both silver and IONPs, silver NPs showed a higher antioxidant activity than IONPs syn. from fenugreek seeds extract [20].
When Alavi and Karimi compared the total antioxidant ability of IONPs, ascorbic acid, and the aqueous leaf extract of A. haussknechtii-synthesized IONPs, green-based IONPs exhibited the least antioxidant activity, followed by ascorbic acid, and the best was the leaf extract [29].
Therefore, we can say that plant-based IONPs synthesis showed moderate antioxidant activity in most of the studies in comparison with the plant aqueous extract, itself, used in their synthesis. This is due to the presence of different functional groups attached to IONPs surface, first, affecting its ability to react with ABTS radicals, second, due to the radicals' stereo selectivity [13].
In addition, the small size of NPs and phytochemicals adsorbed on its surface (flavonoids, polyphenols, terpenoids, etc.), were responsible partially for the high, IONPs formed, antioxidant activity [20]. However, using ultrasonication for IONPs preparation resulted, in an even smaller particles' size and thus, better antioxidant activity [14].
Plant-based green-synthesized IONPs in vitro toxicity studies listed in Table 4  The cytotoxicity of green-synthesized IONPs was tested on WEHI164 fibro sarcoma cells, with no reported significant toxic effects up to the higher concentrations [44], which is a promising clue for their use in the biotechnology field, for drug delivery, and the other mentioned biomedical applications. The green-synthesized IONPs were not reactive to normal cell lines derived from human colon and kidney; CCD112 and HEK293, respectively, as well as to MCF7 for breast cancer, and the lung cancer cell lines (A549) [21,43]. Greensynthesized IONPs also showed no toxic effects towards macrophages upon incubation with IONPs different concentrations for longer periods of time up to 72 h [10,45].
One study evaluated the in vivo toxicity, with no significant changes in the biochemical parameters investigated, between the experimental and control groups [42]. Another study investigated activity for iron deficiency anemia [46]. Four studies have investigated the in vivo hemolytic activity of the green-synthesized IONPs and confirmed no hemolytic activity at lower concentrations [7, 11,22,34].
It is worth mentioning that toxicity is related to either IONPs size or shape, where, the rod-shaped particles showed higher cytotoxicity when compared to the spherical-shaped particles. Six studies mainly presented synthesis, characterization, and reactivity of IONPs without testing their biomedical applications, as shown in Table 6.
Limitation. IONPs synthesized by magnetotactic bacteria, not included in the search, but are among the IONPs with the most remarkable physicochemical properties being superior from the point of view of crystallinity and magnetic properties to many IONPs obtained by chemical conventional methods. Second, the metrics of the green chemistry are not included, as the current review is a systematic rapid brief review, without metaanalysis as for Tobiszewski et al.

Conclusions
Plant-based IONPs green synthesis is now evidenced to be relatively safer, sustainable, and of the least reported or no reported toxicity, when being compared to IONPs synthesized with the conventional chemical methods. Moreover, plant-based IONPs size and properties are superior to NPs synthesized by other methods. Together with the better chemical nature of the plant-based synthesis, IONPs are showing good reliable biomedical properties, namely, antimicrobial (antibacterial/anti-fungal) and anticancer effects.
Proteins, alkaloids, amino acids, alcoholic compounds, polyphenols (catechin, flavones, and phenolic acids), polysaccharides, organic acids, quinones, and other low molecular weight compounds, among sugars, terpenoids, polyphenols, alkaloids, phenolic acids, and proteins have all been implicated in the reduction of the iron ions into IONPs and in promoting their subsequent stability.
Future prospective is to address genetically engineered organisms for IONPs synthesis, with more biomedical applications.