Influence of Physicochemical Properties of Iron Oxide Nanoparticles on Their Antibacterial Activity

Abstract

The increasing occurrence of infectious diseases caused by antimicrobial resistance organisms urged the necessity to develop more potent, selective, and safe antimicrobial agents. The unique magnetic and tunable properties of iron oxide nanoparticles (IONPs) make them a promising candidate for different theragnostic applications, including antimicrobial agents. Though IONPs act as a nonspecific antimicrobial agent, their antimicrobial activities are directly or indirectly linked with their synthesis methods, synthesizing precursors, size, shapes, concentration, and surface modifications. Alteration of these parameters could accelerate or decelerate the production of reactive oxygen species (ROS). An increase in ROS role production disrupts bacterial cell walls, cell membranes, alters major biomolecules (e.g., lipids, proteins, nucleic acids), and affects metabolic processes (e.g., Krebs cycle, fatty acid synthesis, ATP synthesis, glycolysis, and mitophagy). In this review, we will investigate the antibacterial activity of bare and surface-modified IONPs and the influence of physiochemical parameters on their antibacterial activity. Additionally, we will report the potential mechanism of IONPs’ action in driving this antimicrobial activity.

1. Introduction

Antimicrobial resistance (AMR) is a natural phenomenon in which pathogenic microorganisms develop mechanisms to resist antimicrobial drugs.¹ Due to the severity of AMR, it has become a worldwide concern owing to its correlation with escalated morbidity and mortality. According to the World Health Organization (WHO), AMR has been identified as a global threat to public health, and this era has been reported as the “postantibiotic” era, in which minor infections will be the primary cause of death.² According to a study published in 2019, approximately 7.7 million fatalities were ascribed to bacterial infections caused explicitly by antibiotic-resistant bacterial strains.³ Similarly, a report released in 2022 highlighted that AMR was the primary cause of 1.27 million fatalities and had an indirect role in 4.95 million deaths.⁴ Moreover, bacteria develop AMR mechanisms through unsupervised exposure and the wide availability of antibiotics.^5,6 Considering the limited therapeutic options and the severity of AMR, researchers are developing magnetic nanoparticles (MNPs)-based alternative antibacterial agents.⁷⁻⁹

Recently, MNPs have received attention in various fields due to their unique magnetic properties, including paramagnetism, tunable magnetism, biocompatibility, drug delivery, and antimicrobial and surface modification properties.¹⁰⁻¹⁶ Like other MNPs, iron oxide nanoparticles (IONPs) are used as magnetic resonance imaging (MRI) contrast dye, biosensors, diagnosing diseases, drug delivery vehicles, pollutant removal, biomedical machinery, and antimicrobial agents.¹⁷⁻²² Though divalent metals like iron ions are essential for the growth of microbes, IONPs display nonspecific antibacterial activity by generating electrostatic attraction or repulsion force and reactive oxygen species (ROS).²³⁻²⁶ Moreover, other parameters could influence the antimicrobial activity of IONPs, including synthesis methods, precursors, size, and concentration.²⁷ Furthermore, depending on the characteristics of the materials, the physicochemical, electrical, optical, and biological properties of IONPs can be influenced via surface modification. Notably, the coated nanocomplexes exhibit more potent antimicrobial activity than bare IONPs.²⁸

Gudkov et al.²⁹ reported that the antibacterial activity of IONPs could be influenced by altering their synthesis methods and size and discussed several bacteriostatic mechanisms including ROS generation, electrostatic interaction, disruption of the cell membrane, and fragmentation of DNA and protein molecules by inducing free radicals. Moreover, Arias et al.³⁰ described numerous clinical applications of IONPs (e.g., antimicrobial, anticancer, drug delivery, anticonvulsants, and immunosuppressive agents), toxic effects, and the extent of biocompatibility between IONPs and eukaryotic cells or tissues. Another review addressed the biocompatibility and cytotoxicity of IONPs and how their antibacterial activity was evaluated on a various model organisms using multiple physical and chemical criteria.³¹ Alprol et al.³² highlighted that IONPs synthesized through the green methods are able to exhibit potent antibacterial activity against Gram-positive and moderate inhibition against Gram-negative bacteria by inducing oxidative stress by generating ROS. Furthermore, these authors summarized a few influential parameters associated with the antibacterial efficacy of the IONPs and highlighted their mechanisms against various types of bacteria. Recently, Tasnim et al.³³ discussed the antimicrobial mechanisms and activity of several metal-doped IONPs against a range of bacteria and the potential drawbacks of metal-doped IONPs. Nevertheless, the aforementioned reviews were unable to report the influence of diverse coating materials on the physiochemical properties of IONPs, a comparative analysis of the antibacterial activities of the uncoated and surface-coated IONPs-complexes, their combined mechanistic understanding, and the impact of several parameters like shape, concentration, and precursors on the antibacterial activities of IONPs. Therefore, enlightening the influential parameters of the antibacterial activities of the coated and uncoated IONPs should be weighed up for further research purposes, molecular analysis, and drug development.

In this review, we intend to scrutinize the antimicrobial activities of the uncoated and coated IONPs associated with their physicochemical parameters and their potential mechanisms of action(s). Additionally, we will discuss the antimicrobial efficacy of IONPs concerning their precursors, manufacturing techniques, size, shape, concentration, and surface modification materials.

2. Pathogenic Bacteria

Pathogenic bacteria are microorganisms that induce infectious diseases within the host organism.³⁴ They exhibit a diverse array of virulence determinants encompassing adherence factors, invasion factors, toxins, capsules, and siderophores, thereby eliciting pathogenicity and instigating disease through host cell invasion and evasion of host defense mechanisms.^35,36

According to the structural composition of the bacterial cell wall, bacteria can be classified into two distinct categories: Gram-positive and Gram-negative.³⁷ Several studies have shown that a significant proportion of pathogenic bacteria exhibit Gram-negative characteristics, and due to their distinctive cellular structure, they are more susceptible to antibiotic resistance.³⁸ Gram-negative bacteria have a complex cellular structure consisting of a peptidoglycan cell wall surrounded by the lipopolysaccharide (LPS) layer. The LPS comprises several virulence factors, including Lipid-A, also known as endotoxin, which is the main virulence factor that causes disease.³⁹ Gram-negative bacteria release their virulence factor to cause disease during active cellular growth, and after the cell has been lysed, endotoxin is released.⁴⁰ Upon releasing these toxins, a robust inflammatory response is initiated within the host organism, potentially resulting in severe and life-threatening symptoms.^41,42 Multiple toxins can potentially inflict diverse harm on the host cell. For instance, producing Shiga toxin by E. coli and releasing cholera toxin (CT) by V. cholerae can lead to severe watery diarrhea.⁴³ The prevailing ailments resulting from pathogenic bacteria include tuberculosis caused by Mycobacterium tuberculosis,⁴⁴ stomach infections caused by H. pylori,⁴⁵ and typhoid fever caused by Salmonella typhi.(46) More importantly, pathogenic bacteria are able to infiltrate the host’s tissue and employ various strategies to exploit the host, potentially resulting in adverse health outcomes and a diverse array of illnesses.⁴⁷ Pathogen invasion necessitates a designated point of entry originating from a contaminated source, such as consumable items or inhaling noxious substances. Moreover, pathogens can be transmitted through direct and indirect contact with an infected individual or in poorly ventilated environments.⁴⁸

Pathogenic bacteria pose a significant challenge with the emergence of antibiotic-resistant strains. The overutilization of antibiotics in bacterial populations leads to genetic mutations or the expression of resistance genes, resulting in antibiotic-resistant bacteria development.⁴⁹ Resistant genes can be transferred to other bacteria through gene transfer, which may occur through many processes, including conjugation, transmission, and transduction. When pathogenic bacteria develop antibiotic resistance, they become significantly more challenging to treat and present an increased risk to the host organism.⁵⁰

3. Iron Oxide Nanoparticles (IONPs)

Ferromagnetic IONPs find extensive application due to their intricate structure. IONPs exhibit either spinel or inverse spinel crystalline structures, which are determined by the specific type of IONPs and their underlying condition of synthesis.⁵¹ Iron cations and oxide ions are arranged in tetrahedral and octahedral sites.^52,53 However, the positioning of the iron ions is influenced by their oxidation state and structure.⁵⁴ Fe³⁺ ions prefer occupying octahedral and tetrahedral sites while maintaining their high spin complex, which arises from their ability to polarize water molecules, leading to the facile cleavage of OH bonds. This phenomenon gives rise to a complex with a reduced charge density and increased stability, accompanied by a smaller ionic radius. In contrast, it was observed that Fe²⁺ exhibits a preference for occupying exclusively octahedral positions within a complex. Moreover, the preferred attributes of the lower charge density and wider ionic radius of Fe²⁺ enable them to maintain a high spin configuration.⁵⁵

IONPs have 14 different types of structures, and 10 of them are found in the Earth’s crust due to the abundant existence of oxygen, hydrogen, and iron. Among them, the predominant rock-forming minerals are hematite, magnetite, and goethite. Maghemite, ferrihydrite, and lepidocrocite have a moderate abundance, whereas wustite, bernalite, and feroxyhyte are the least abundant. Hematite (Fe₂O₃) and magnetite (Fe₃O₄) have been extensively studied and applied in different clinical and biomedical domains due to their distinctive attributes such as ferromagnetism, biocompatibility, adjustable magnetic properties, coercivity, high magnetic saturation, magneto-optic performance, anisotropy, magneto-crystalline anisotropy, surface modification properties, and photothermal properties.⁵⁶⁻⁶¹Figure 1 shows the schematical representation of the most abundant IONPs and oxyhydroxides.

Figure 1.

Schematic representation of (a) hematite, (b) maghemite, (c) magnetite, (d) lepidocrocite, and (e) goethite structures. These structures are viewed from the ⟨001⟩ or ⟨0001⟩ directions. The idea of the diagram is taken from Guo and Barnard.⁸²

There are multiple methodologies for synthesizing IONPs, which can broadly be categorized into two fundamental approaches. The top-down approach involves producing NPs from larger particles.⁶² A different approach involves using the bottom-up method, where NPs are created by utilizing individual molecules and gradually assembling them into the desired nanostructure.⁶³⁻⁶⁵ The methodologies employed in this study encompass coprecipitation (COP), sol–gel (SG), hydrothermal (HDT), one-pot (OP), green synthesis (GRS), sonochemical (SNC), laser pyrolysis (LP), solid-state pyrolytic (SSP), polyol synthesis (PYS), and the microemulsion method (MEL).^17,66−75 Utilizing these synthesis methods significantly influences various characteristics of the IONPs, including their size, cation distribution, magnetic properties,¹⁰ and antimicrobial properties.⁷⁶ Moreover, the versatile synthesis process can significantly influence the physicochemical properties of IONPs, which have found widespread applications, including contrast dyes for MRI, specific drug delivery agents, gene carriers for gene therapy, potential antimicrobial agents, potential cancer therapy agents, and biosensors.⁷⁷⁻⁷⁹ Besides drug delivery, IONPs could enhance the efficacy of Food and Drug Administration (FDA)-approved drugs.^80,81

4. IONPs as an Antimicrobial Agent

IONPs possess unique antibacterial properties due to their distinctive physical and chemical attributes, such as their surface charge and ability to generate ROS.⁸³ The features of IONPs depend on various aspects, such as their synthesis process, precursors, size, shape, and concentration.^9,84

IONPs could be synthesized using various techniques, including COP, SG, HDT, GRS, LP, MEL, OP, SNC, and PSY methods.⁸⁵ Chemically synthesized methods necessitate the use of intense radiation and result in the production of very hazardous residues. The GRS method requires minimal precursors and energy for initiation, with plant extracts as the reducing agent. Interestingly, the plant extracts possess inherent therapeutic characteristics that contribute to their notable antibacterial efficacy.^85,86 Moreover, various iron precursors utilized in the synthesis procedures may influence the properties of IONPs (Table 1). Hence, precursors and synthesis methods are considered crucial parameters that influence the antibacterial activity of IONPs.

Table 1. Antimicrobial activity and potential mechanism of chemically synthesized IONPs^a.

NPs	Particle size (nm)	Shape	Precursor	Synthesis method	Coating Material	Con. (μg/mL)	Organism	ZOI (mm)	MIC (μg/mL)	MBC (μg/mL)	IC50 (μg/mL)	Mechanism	ref.
Fe3O4	∼254	Spherical	FeCl3·6H2O, FeCl2·4H2O, NaOH, DW & HCl	COP	RHL	NR	Escherichia coli (O157: H7)	∼9	64	128	NR	The RHL coated-Fe3O4@PVA@p-CoA/GA biosurfactant mediated the structural and confirmational changes on the cell surface. Moreover, the coating material intercalated with the DNA, inhibited the replication, transcription and translation process by generating ROS. The ROS induced oxidative stress that led to apoptosis of bacterial cells.	(87)
							Escherichia coli (O26: H11)	∼10	4	8
							Escherichia coli (O78: H10)	∼12	32	64
							Staphylococcus aureus (MSSA)	∼12	16	32
							Staphylococcus aureus (MRSA)	∼10	32	64
							Staphylococcus aureus (VRSA)	∼8	64	128
					RHL, PVA, GA, p-CoA	NR	Escherichia coli (O157: H7)	35	32	64
							Escherichia coli (O26: H11)	45	1	2
							Escherichia coli (O78: H10)	40	16	32
							MSSA	40	4	8
							MRSA	30	16	32
							VRSA	25	32	64
Fe2O3	36.1	Spherical	FeCl3·6H2O, TiCl4·2H2O, CTAB & NaOH	COP	PR	5000	Bacillus subtilis	13	NR	NR	NR	The Fe2O3 and TiO2 polyester nanocomposite enhanced the interaction with bacterial cell membranes. It created an ionic imbalance in the bacterial cell, resulting in cell apoptosis.	(88)
							Staphylococcus aureus	18
							Escherichia coli	13
							Pseudomonas aeruginosa	12
							Candida albicans	29
							Aspergillus niger	26
Fe3O4	48	Spherical	FeSO4, FeCl3, DW, NH4OH & HNO3,	COP	RHL	NR	Pseudomonas aeruginosa	NR	1000	NR	NR	Fe3O4 induced oxidative stress in the bacterial cell by producing ROS which caused cell death. Again, the Rhamnolipid-coated Fe3O4 interacted with extracellular polymeric substances (EPS), which could affect the hydrophobicity of bacterial cell surface and hinder biofilm generation.	(89)
							Staphylococcus aureus	NR	1000	NR
IO QDNPs	<80	Spherical	Fe (NO3)3.9H2O, argon gas, DW, ethanol & NaOH	COP and HDT.	NR	NR	Escherichia coli PTCC 1330	NR	1	NR	NR	The IO-QDNPs changed membrane potential and damaged the biological mechanism by interacting with the cell membranes and subunit of the ribosome. Again, ROS-mediated structural and confirmational changes in DNA were responsible for bacterial cell death.	(90)
							Staphylococcus aureus PTCC 1053	NR	0.5
							Serratia marcescens PTCC1621	NR	0.5
							Pseudomonas aeruginosa PTCC1074	NR	0.5
							Staphylococcus aureus PTCC. 1112	NR	1
							Micrococcus luteus PTCC. 1110	NR	4
							Bacillus subtilis PTCC. 1023	NR	0.5
							Staphylococcus epidermidis PTCC 1114	NR	0.5
Fe3O4	∼39.56	Spherical	FeCl2·4H2O, FeCl3·6H2O & NH4OH	COP	TiO2	NR	Escherichia coli	NR	150	NR	NR	TiO2-coated IONPs had potent toxicity against bacterial cells, so they exhibited antibacterial activity when they interacted with the cell membranes.	(91)
							Klebsiella pneumonia	NR	150
							Bacillus subtilis	NR	150
							Staphylococcus aureus	NR	150
							Escherichia coli	NR	150
							Klebsiella pneumonia	NR	150
							Bacillus subtilis	NR	150
							Staphylococcus aureus	NR	150
SPION	23.32 ± 1.17	NR	Iron salt and dimercaptosuccinic acid	Two-step COP process.	NR	NR	MARS	NR	530.796 ± 15.241	NR	NR	SPION mediated the conformational and structural changes on the proteins of the cell surface and disrupted the cell membrane.	(92)
									495.993 ± 3.909	NR
									581.353 ± 10.26	NR
									457.871 ± 21.064	NR
Fe3O4	31.09	Spherical	FeCl2·2H2O, FeCl3·6H2O, N2, DW & NH4OH.	COP	NR	NR	Staphylococcus epidermidis	17.33 ± 0.57	25	50	NR	Generally, photolytic generation of ROS induces oxidative stress in the cell. Also, due to the vibration of magnetic fields, IONPs lead bacterial cells to death.	(93)
							Proteus mirabilis	17.51 ± 0.57	50	100
							Acinetobacter baumannii	17.66 ± 0.57	50	100
					PEG	NR	Staphylococcus epidermidis	19.66 ± 0.57	25	50
							Proteus mirabilis	21.66 ± 0.57	50	100
							Acinetobacter baumannii	22.00 ± 0.46	50	100
					GEN	NR	Staphylococcus epidermidis	21.33 ± 1.15	50	100
							Proteus mirabilis	23.66 ± 0.57	25	50
							Acinetobacter baumannii	23.66 ± 0.57	50	100
					PEG+ GEN	NR	Staphylococcus epidermidis	23.66 ± 0.057	50	100
							Proteus mirabilis	25.66 ± 0.57	25	50
							Acinetobacter baumannii	26.33 ± 0.57	50	100
Fe2O3	14.2 ± 0.5	Spherical	FeCl2·4H2O, FeCl3·6H2O, DW, NH4OH, HNO3, HCl & Fe (NO3)3·9H2O	COP	Teicoplanin	NR	Bacillus subtilis (ATCC 6633)	NR	2	>128	NR	Fe2O3 NPs induced ROS, which induced oxidative stress and led to programmed cell death as well as autophagic activity.	(94)
							Staphylococcus aureus (ATCC 6538Pb (MSSA))	NR	2	128
							Staphylococcus aureus (ATCC 43300 (MRSA)	NR	2	>128
							Enterococcus. faecalis (ATCC 29212)	NR	1	32
							Enterococcus. faecalis (ATCC 51299 (VanB)	NR	2	>128
							Enterococcus. faecalis (9160188401-EF-34 (VanA)	NR	>128	>128
							Escherichia coli (ATCC 35218)	NR	>128	>128
Fe2O3	25.34	Hexagonal	FeSO4·7H2O, Co (NO3)2·6H2O, NaOH & DW	COP	NR	400	Bacillus subtilis	11	Escherichia coli			Fe2O3 NPs induced ROS, which triggered oxidative stress and led to cell death.	(59)
							Staphylococcus aureus	12	900	NR
							Escherichia coli	19
							Salmonella typhi	12
						600	Bacillus subtilis	15
							Staphylococcus aureus	13
							Escherichia coli	20
							Salmonella typhi	13
						800	Bacillus subtilis	17
							Staphylococcus aureus	15
							Escherichia coli	21
							Salmonella typhi	16
Fe3O4	10.64 ± 4.73	Spherical	FeSO4·7H2O, FeCl3·6 H2O, DW & NaOH	COP	Oleic acid	NR	Escherichia coli	NR	NR	NR	NR	The antibacterial activity of Fe3O4 NPs was mediated by electrostatic interaction between NPs and the cell membranes. Also, the ROS played a vital role in cell apoptosis.	(95)
							Enterococcus hirae	NR
Fe3O4	10–30	Spherical	NR	COP	NR	12.5	Escherichia coli	7	NR	NR	NR	Fe3O4 mediated antibacterial activity by causing damaged to the proteins and DNA in the bacterial cell by generating ROS.	(96)
							Proteus mirabilis	7
							Bacillus subtills	7
						25	Escherichia coli	7
							Proteus mirabilis	7
							Bacillus subtills	7
						50	Escherichia coli	8
							Proteus mirabilis	8
							Bacillus subtills	8
						Fe3O4 + Plant extract
						12.5	Escherichia coli	11	NR	NR	NR
							Proteus mirabilis	16
							Bacillus subtills	7
						25	Escherichia coli	12
							Proteus mirabilis	17
							Bacillus subtills	8
						50	Escherichia coli	13
							Proteus mirabilis	18
							Bacillus subtills	10
Fe3O4	6–9	Spherical	FeCl3, FeSO4, DW, NH4OH, PEG & ethyl alcohol	COP	NR	NR	Serratia marcescens	NR	32	NR	NR	ROS produced by IONPs moderated physical damage or chemical damage by interacting with the cell membranes, leading to bacterial cell death.	(97)
							Escherichia coli	NR	64
							Pseudomonas aeruginosa	NR	128
							Listeria monocytogenes	NR	32
Fe3O4	24	NR	FeCl3, FeCl2, DW, Argon gas & NaOH	COP	NR	40	Bacillus cereus	13	5	80	NR	IONPs could interact with DNA and proteins and mediate their conformational changes.	(66)
							Klebsiella pneumoniae	15
						80	Bacillus cereus	22
							Klebsiella pneumoniae	26
Fe3O4	10–14	Spherical	FeCl3, FeCl2, DW, & NaOH	COP	NR	500	Staphylococcus aureus	<10	Staphylococcus aureus		NR	The IONPs damaged the cell membrane by inducing ROS. When IONPs are conjugated with gentamicin, bacterial growth is constrained by both mechanisms. By inhibiting the protein synthesis and damaging the cell membrane.	(98)
							Escherichia coli	<10	25,000	30,000
							Bacillus subtills	<10
							Pseudomonas aeruginosa	<10
						1000	Staphylococcus aureus	<10	Escherichia coli		NR
							Escherichia coli	<10	28,000	35,000
							Bacillus subtills	<10
							Pseudomonas aeruginosa	<10
						3000	Staphylococcus aureus	<10	Bacillus subtills		NR
							Escherichia coli	<10	20,000	25,000
							Bacillus subtills	<10
							Pseudomonas aeruginosa	<10
						5000	Staphylococcus aureus	<10	Pseudomonas aeruginosa		NR
							Escherichia coli	<10	30,000	35,000
							Bacillus subtills	<10
							Pseudomonas aeruginosa	<10
					GEN	500	Staphylococcus aureus	<10	Staphylococcus aureus		NR
							Escherichia coli	<10	2000	3000
							Bacillus subtills	12.5
							Pseudomonas aeruginosa	<10
						1000	Staphylococcus aureus	10	Escherichia coli		NR
							Escherichia coli	<10	2500	3000
							Bacillus subtills	13
							Pseudomonas aeruginosa	<10
						3000	Staphylococcus aureus	13	Bacillus subtills		NR
							Escherichia coli	<10	1500	2500
							Bacillus subtills	25
							Pseudomonas aeruginosa	<10
						5000	Staphylococcus aureus	16	Pseudomonas aeruginosa		NR
							Escherichia coli	11	3000	4000
							Bacillus subtills	33
							Pseudomonas aeruginosa	<10
Fe3O4	∼100	Spherical	Oleic amine, ethylene glycol, FeCl3·6H2O, & sodium acetate	HDT	NR	200	Klebsiella pneumoniae	22 ± 1.0	Klebsiella pneumoniae		NR	IONPs generate ROS molecules that interact with the ion transportation channel and the DNA and induce oxidative stress to eliminate bacterial cells.	(68)
							Staphylococcus aureus	28 ± 0.5	50	100
							Enterococcus faecalis	25 ± 0.0
							Pseudomonas aureginosa	18 ± 1.0
						100	Klebsiella pneumoniae	20. ± 0.0	Staphylococcus aureus
							Staphylococcus aureus	25 ± 0.0	6.25	12.5
							Enterococcus faecalis	20 ± 1.0
							Pseudomonas aureginosa	16 ± 0.0
						50	Klebsiella pneumoniae	18 ± 1.0	Enterococcus faecalis
							Staphylococcus aureus	22 ± 2.0	6.25	12.5
							Enterococcus faecalis	17 ± 0.5
							Pseudomonas aureginosa	15 ± 0.5
						25	Klebsiella pneumoniae	15 ± 0	Pseudomonas aureginosa
							Staphylococcus aureus	20 ± 1.0	50	50
							Enterococcus faecalis	11 ± 0.0
							Pseudomonas aureginosa	12 ± 0.0
Fe3O4	35	Spherical	Fe (NO3)3·9H2O, DW, & NH3	CC	NR	25	Escherichia coli	15	NR	NR	NR	Fe3O4 generated ROS that disrupted membrane protein and penetrated the bacterial membrane. It induced oxidative stress, which led to cell death.	(99)
							Proteus vulgaris	11
							Staphylococcus aureus	9
							Xanthomonas	10
						30	Escherichia coli	16
							Proteus vulgaris	12
							Staphylococcus aureus	12
							Xanthomonas	12
						40	Escherichia coli	17
							Proteus vulgaris	15
							Staphylococcus aureus	14
							Xanthomonas	14
						50	Escherichia coli	17
							Proteus vulgaris	20
							Staphylococcus aureus	15
							Xanthomonas	15
						60	Escherichia coli	15
							Proteus vulgaris	20
							Staphylococcus aureus	11
							Xanthomonas	8
						80	Escherichia coli	15
							Proteus vulgaris	12
							Staphylococcus aureus	12
							Xanthomonas	14
						100	Escherichia coli	21
							Proteus vulgaris	21
							Staphylococcus aureus	15
							Xanthomonas	15
IO	14–23	Hexagonal	FeSO4·7H2O, β-Cyclodextrin, N2 & sodium borohydride	CS	Beta-cyclodextrin	NR	Staphylococcus aureus	43.83 ± 0.75	100	NR	NR	IONPs interacted with the cell wall and facilitated cell permeability, hindering cellular activity and resulting in the disruption of bacterial cells.	(100)
							Klebsiella pneumonia	39.83 ± 1.33	300
							Salmonella typhi	20 ± 0.63	200
Fe3O4	∼25	NR	Fe (NO3)3, DW, Ethylene glycol.	SG	NR	5	Bacillus spp.	23	NR	NR	NR	The addition of citric acid increased the ROS generation and the electro-hole pair generation, leading to the decay of the bacterial surface.	(101)
							Escherichia coli	19
						10	Bacillus spp.	25
							Escherichia coli	20
						15	Bacillus spp.	26
							Escherichia coli	21
						20	Bacillus spp.	27
							Escherichia coli	22
					Citric acid	5	Bacillus spp.	32
							Escherichia coli	29
						10	Bacillus spp.	31
							Escherichia coli	22
						15	Bacillus spp.	33
							Escherichia coli	23
						20	Bacillus spp.	36
							Escherichia coli	26
Fe3O4	NR	NR	Fe (NO3)3·9H2O, ethanol	SG	NR	50	Enterobacter aerogenes	NR	NR	NR	NR	Fe3+ ions interact with the negatively charged membrane, decreasing the membrane integrity. By entering the cell, IONPs produce ROS, which disrupts the cell organelles and eradicates the cell.	(102)
						100
						150	Staphylococcus aureus
						200
Fe2O3	50–110	Spherical	SDS, DMF & iron pressed pellet.	PLA	NR	Fe2O3 + DMF						The electrons in the photocatalytic process were absorbed by free radicals and produced OH– radicals. These radicals inhibited the growth of bacteria by induced oxidative stress generated by ROS.	(103)
						4250 (20 mJ)	Escherichia coli	19	NR	NR	NR
							Pseudomonas aeruginosa	17
							Serritia marcescens	20
							Staphylococcus aureus	21
						4250 (40 mJ)	Escherichia coli	22
							Pseudomonas aeruginosa	16
							Serritia marcescens	24
							Staphylococcus aureus	18
						4250 (60 mJ)	Escherichia coli	18
							Pseudomonas aeruginosa	27
							Serritia marcescens	17
							Staphylococcus aureus	22
						4250 (80 mJ)	Escherichia coli	17
							Pseudomonas aeruginosa	27
							Serritia marcescens	20
							Staphylococcus aureus	23
						Fe2O3 + SDS
						4250 (20 mJ)	Escherichia coli	25
							Pseudomonas aeruginosa	13
							Serritia marcescens	27
							Staphylococcus aureus	30
						4250 (40 mJ)	Escherichia coli	27
							Pseudomonas aeruginosa	27
							Serritia marcescens	30
							Staphylococcus aureus	35
						4250 (60 mJ)	Escherichia coli	23
							Pseudomonas aeruginosa	22
							Serritia marcescens	15
							Staphylococcus aureus	28
						4250 (80 mJ)	Escherichia coli	20
							Pseudomonas aeruginosa	19
							Serritia marcescens	18
							Staphylococcus aureus	30

^aNPs: Nanoparticles; Con.: Concentration; ZOI: zone of inhibition; ref: References; ROS: Reactive oxygen species; NR: Not reported; MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration; IC₅₀: Half maximal inhibitory concentration; COP: Coprecipitation; HDT: Hydrothermal; CS: Chemical synthesis; SG: Sol–gel; TDP: Thermal decomposition; PLA: Pulsed laser ablation; FeCl₃·6H₂O: Ferric chloride hexahydrate; FeCl₂·4H₂O: Ferrous chloride tetrahydrate; FeSO₄: Ferrous sulfate; TiCl₄·2H₂O: Titanium tetrachloride; FeSO₄·7H₂O: Ferrous sulfate; Fe (NO₃)₃·9H₂O: Ferric nitrate nonahydrate; DW: Deionized water; CTAB: Trimethylammonium bromide; PR: polyester resin; RHL: Rhamnolipid; DMF: Dimethylformamide; GEN: Gentamicin; kan: Kanamycin; CC: Chemical combustion; PEG: Poly(ethylene glycol); IO: Iron oxide; QNDPs: Quantum dots nanoparticles; SPION: Superparamagnetic Iron oxide nanoparticles; PVA: Poly(vinyl alcohol) polymer; GA: Gallic acid; p-CoA: p-Coumaric acid; RHL coated-Fe₃O₄@PVA@p-CoA/GA: Rhamnolipid coated Iron oxide with Poly(vinyl alcohol) polymer; Gallic acid & p-Coumaric acid; MSSA: Methicillin-sensitive Staphylococcus aureus; MRSA: Methicillin-resistant Staphylococcus aureus; VRSA: Vancomycin-resistant Staphylococcus aureus.

The antimicrobial efficacy of IONPs also varies by size, which influences the surface area-to-volume ratio. The small size of the IONPs indicates a larger surface area to volume ratio; consequently, the increased surface area would allow IONPs to cover a more significant portion of the bacteria, resulting in more frequent interactions with the bacterial cell membrane and causing damage through electrostatic attraction and repulsion force.^104−106 The antibacterial effectiveness of the IONPs also depends on the bacterial strains due to their differences in cell wall structure. The Gram-positive bacteria have a thick peptidoglycan layer consisting of a net negative charge due to teichoic acid.¹⁰⁷ On the contrary, Gram-negative bacteria consist of a thin peptidoglycan layer and an overlaid lipopolysaccharide layer, which has an elevated net negative charge. IONPs interact with the negatively charged bacterial cell membrane through an electrostatic force that induces depolarization and disrupts the integrity of the cell membrane.^103,108,109 Hence, due to the structural difference in the cellular composition, Gram-positive bacteria are more susceptible to IONPs than Gram-negative bacteria. Moreover, IONPs implicate nonspecific mechanistic behavior to eradicate pathogenic bacteria, such as inducing oxidative stress by producing ROS.^110−112 These have the ability to inhibit the function of DNA, proteins, and enzymes, leading to bacterial cell death.^76,113 IONPs do not adhere to a single strategy for inhibiting bacteria. As a result, bacteria struggle to develop resistance mechanisms against them.¹¹⁴

5. Parameters Influencing Antimicrobial Activity

5.1. Synthesis

5.1.1. COP Method

COP is the most common, economical, and versatile method for generating IONPs. This method generates IONPs under alkaline conditions through precipitation in a nonoxidizing environment where Fe²⁺/Fe³⁺ salt precursors are mixed in a 1:2 ratio.^96,115−117 Moreover, the COP technique can effectively influence the size and morphology of the IONPs by altering some variables during synthesis, including the ionic strength, pH, temperature, concentration, and ratio of the precursors.¹¹⁸

IONPs synthesized via the COP method exhibited notable antibacterial activity against bacteria; for instance, the bare Fe₃O₄ exhibited moderate antibacterial activity against E. coli, P. mirabilis, and B. subtilis.(96) However, the antibacterial activity can be increased through surface modification of IONPs. Recently, Sharaf et al.⁸⁷ reported that IONPs generated and modified using COP procedures showed notable antibacterial efficacy against several E. coli and S. aureus. An inhibitory zone ranging from 9 to 12 mm was detected against both bacteria when the Fe₃O₄ was coated with RHL. Interestingly, surface modification of the IONPs using RHL, PVA, GA, and p-CoA caused a 4-fold increase in zone of inhibition (ZOI). Conversely, the Fe₂O₃ generated by COP without other substances demonstrated an enhanced antibacterial effect compared to magnetite. It displayed a ZOI ranging from 11 to 21 mm against B. cereus, S. aureus, E. coli, and S. typhi at greater concentrations.⁵⁹ Compared to bare Fe₃O₄ NPs, the Fe₂O₃ NPs showed pronounced bacteriostatic action against E. coli and S. aureus,^59,98 while a study by Abdulsada et al.⁹³ displayed that IONPs synthesized and modified with poly(ethylene glycol) (PEG) and gentamicin (GEN) through COP had an elevated antibacterial activity compared to the bare IONPs. Despite having the same synthesis method, IONPs exhibit a varying response against identical bacteria, and several factors may contribute to this variation, including precursors, shape, size, and coating material. Thus, IONPs synthesized by the COP method exhibited remarkable antimicrobial properties.⁹⁹

5.1.2. SG Method

The SG method is a bottom-up synthesis method where irreversible chemical reactions occur. Certain chemical interactions, including hydrolysis and condensation, form a sol and a three-dimensional polymeric structure termed a gel. The polycondensation reaction generates a polymeric network that IONPs can retain. In most cases, nitrate precursors are utilized in SG methods for several key benefits, including uniformity in size and high levels of purity and quantity for IONPs.¹¹⁹ For these reasons, IONPs produced through the SG method exhibit noteworthy antibacterial activity against various bacteria at varying concentrations. The SG method is widely utilized to synthesize doped metal IONPs; however, some research indicates the synthesis of IONPs through the SG method and observes their antibacterial activity against some Gram-negative and positive bacteria. For instance, Fe₃O₄ was generated through the SG procedure, and its antibacterial activity was observed at different concentrations ranging from 5 to 20 μg/mL on different Gram-negative and positive bacteria. IONPs at different concentrations exhibited 19 to 36 mm ZOI for E. coli and B. cereus(101) (Table 2). Additionally, Brahmbhatt et al.¹⁰² conducted an antibiogram with Fe₃O₄ produced by SG and gave a visual aid for ZOI; however, the experiment lacked statistical analysis regarding the antibacterial activity of IONPs.

Table 2. Antimicrobial activity and potential mechanism of Green synthesized IONPs^a.

NPs	Particle size (nm)	Shape	Precursor	Synthesis method	Coating Material	Con. (μg/mL)	Organism	ZOI (mm)	MIC (μg/mL)	MBC (μg/mL)	IC50 (μg/mL)	Mechanism	ref
α-Fe2O3	28.17	Spherical	FeCl3·6H2O, & Capparis zeyl anica leaf extract	GRS	NR	25	Staphylococcus aureus	23 ± 2.23	NR	NR	15.6	Fe2O3 eliminates bacterial cells by inducing oxidative stress by generating ROS molecules.	(140)
							Streptococcus pyogenes	25 ± 2.56
							Escherichia coli	23 ± 1.34
							Pseudomonas aeruginosa	22 ± 1.23
							Candida albicans	19 ± 0.98
							Aspergillus niger	17 ± 1.62
Fe2O3	13.13–24.93	Hexagonal	Purpureocillium lilacinum,& FeSO4	GRS	NR	NR	Staphylococcus aureus	26.5	NR	NR	NR	Fe2O3 mediated structural or conformational change of DNA, which disrupted enzymatic activity by generating hydroxyl free radicals. Additionally, Fe2O3 induced oxidative stress, leading to apoptosis of bacterial cells.	(131)
							Bacillus subtilis	24.8
							Escherichia coli	19.5
							Pseudomonas aeruginosa	17
IO	3.31–10.69	Spherical	FeCl3& Penicillium spp.	GRS	NR	250	Staphylococcus aureus	12 ± 0.6	NR	NR	NR	IONPs diffuse into the intercellular membrane and damage DNA and protein and decompose the lipopolysaccharide layer by generating ROS, which induces oxidative stress. Hence, bacterial growth was inhibited.	(139)
							Escherichia coli	11.3 ± 1.2
							Klebsiella pneumoniae	11.3 ± 0.6
							Shigella sonnie	11.3 ± 0.6
							Pseudomonas aureginosa	11.3 ± 0.6
Fe2O3	40	Spherical	Citrus leaf extract, DW, & FeCl3.	GRS	NR	100	Bacillus subtilis	26.1 ± 0.24	NR	NR	NR	Fe2O3 disrupted the enzymatic activity by interacting with cell surface proteins and the biological metabolites, resulting in bacterial cell apoptosis.	(69)
							Klebsiella pneumoniae	21.5 ± 0.36
						50	Bacillus subtilis	24.1 ± 0.37
							Klebsiella pneumoniae	18.3 ± 0.49
						25	Bacillus subtilis	12.8 ± 0.69
							Klebsiella pneumoniae	11.7 ± 0.53
Fe2O3	5–100	Spherical	FeCl3, Tamarix Aphylla extract& NaOH	′GRS	NR	1000	Escherichia coli	11	30 ± 0.5	NR	NR	Fe2O3 diffused into the cytoplasmic space, disrupted the enzymatic activity, damaged the DNA and led to death.	(141)
							Bacillus subtilis	12	30 ± 0.5
FeO	93.9	Spherical	FeSO4·7H2O Thymbra spicata leaf extract& ethyl alcohol.	GRS	NR		Bacillus cereus	NR	200	200	NR	The free radical generated by FeO NPs interacted with the cellular membrane, which induced oxidative stress and caused cell apoptosis.	(142)
							Staphylococcus aureus		>200	>200
							Escherichia coli		>200	>200
							Salmonella typhimurium		100	200
					NR		Bacillus cereus	NR	200	>200	NR
							Staphylococcus aureus		>200	>200
							Escherichia coli		>200	>200
							Salmonella typhimurium		200	>200
IONPs	150–200	Spherical	FeSO4·7H2O, Lawsonia inermis extract, NaOH, DW & methanol.	GRS	l-tyr	40	Staphylococcus aureus	16	NR	NR	NR	l-tyrosine-coated IONPs interacted with the lipid bilayer, disrupting the bacterial cell membrane and leading to cell apoptosis.	(143)
							Staphylococcus tryphimurium	15
FeO	82 ± 7.0	Spherical	FeCl3, PMC, & DW	GRS	NR	10	Escherichia coli	20	NR	NR	NR	FeO-NPs interacted with the bacteria’s thiol group, which induced oxidative stress and caused cell death.	(144)
							Salmonella typhi	24
							Staphylococcus aureus	26
							Shigella	24
							Pasteurella multocida	18
							Pseudomonas aeruginosa	16
FeO	10–25	Spherical	FeCl3 & GMAM	GRS	NR	NR	Escherichia coli	NR	NR	200	NR	FeO-NPs interacted with bacterial cell membranes and induced oxidative stress by producing ROS, leading to cell death.	(145)
							Pseudomonas aeruginosa	NR	NR	200
							Staphylococcus aureus	NR	NR	200
Fe3O4	84.81	NR	FeCl2, FeCl3, DW, MH, CCP, & NaOH	GRS	Kan	25 μg (NPs) + 5 μg (antibiotic)	Bacillus cereus (ATCC 13601)	10.35 ± 0.17				Antibiotics or anticandidal-coated Fe3O4 adheres to the cell surface via electromagnetic attractions; they interact with the thiol group which induces oxidative stress and leads to microbial cell apoptosis.	(123)
							Escherichia coli (ATCC 43890)	18.44 ± 0.41
							Listeria monocytogenes (ATCC 19115)	16.00 ± 0.86
							Staphylococcus aureus (ATCC 49444)	13.09 ± 0.30
							Salmonella typhimurium (ATCC 43174)	13.17 ± 0.26
					Rif	25 μg (NPs) + 5 μg (antibiotic)	Bacillus cereus (ATCC 13601)	9.36 ± 0.28
							Escherichia coli (ATCC 43890)	9.62 ± 0.51
							Listeria monocytogenes (ATCC 19115)	0
							Staphylococcus aureus (ATCC 49444)	21.99 ± 1.32
							Salmonella typhimurium (ATCC 43174)	0
					Amp	25 μg (NPs) + 5 μg (antibiotic)	Candida albicans (KACC 30003)	10.63 ± 0.55
							Candida albicans (KACC 30062)	17.68 ± 0.52
							Candida glabrata (KBNO6P00368)	10.11 ± 0.20
							Candida glochares (KACC 30061)	17.35 ± 0.35
							Candida saitoana (KACC 41238)	12.30 ± 0.54
	48.91			GRS	Kan	25 μg (NPs) + 5 μg (antibiotic)	Bacillus cereus (ATCC 13601)	10.04 ± 0.41
							Escherichia coli (ATCC 43890)	19.65 ± 0.60
							Listeria monocytogenes (ATCC 19115)	13.10 ± 0.34
							Staphylococcus aureus (ATCC 49444)	12.28 ± 0.15
							Salmonella typhimurium (ATCC 43174)	12.42 ± 0.28
					Rif	25 μg (NPs) + 5 μg (antibiotic)	Bacillus cereus (ATCC 13601)	10.66 ± 0.16
							Escherichia coli (ATCC 43890)	9.29 ± 0.57
							Listeria monocytogenes (ATCC 19115)	0
							Staphylococcus aureus (ATCC 49444)	24.42 ± 0.26
							Salmonella typhimurium (ATCC 43174)	0
					Amp	25 μg (NPs) + 5 μg (antibiotic)	Candida albicans (KACC 30003)	10.33 ± 0.46
							Candida albicans (KACC 30062)	16.15 ± 0.23
							Candida glabrata (KBNO6P00368)	9.81 ± 0.57
							Candida glochares (KACC 30061)	16.44 ± 0.21
							Candida saitoana (KACC 41238)	10.01 ± 0.16
α-Fe2O3	34	Quasi-spherical	FeCl3, P. guajava leaf extracted, DW & acetone	GRS	NR	20	Escherichia coli	29	NR	NR	NR	Fe2O3 produced ROS, which broke the DNA strands, inactivated enzymes and led to lipid peroxidation. This activity triggered mechanisms such as penetration of cell membrane and induced oxidative stress which mediated cell death.	(122)
							Staphylococcus aureus	39
						50	Escherichia coli	10
							Staphylococcus aureus	13
						100	Escherichia coli	12
							Staphylococcus aureus	15
Fe3O4	37.86	Spherical	FeCl3, FeCl2, Zea mays L., & ear leaf extract.	GRS		Fe3O4 (25 μ) + kan (5 μ)	Bacillus cereus ATCC 13061	9.8 ± 0.34	NR	NR	NR	The ROS mediated by Fe3O4 NPs damaged DNA, protein and cellular membranes. Thus, resulted in bacterial cell death	(146)
							Escherichia coli ATCC 43890	18.86 ± 0.82
							Listeria monocytogenes ATCC 19115	13.54 ± 0.30
							Staphylococcus aureus ATCC 49444	13.09 ± 0.15
							Salmonella typhimurium ATCC 43174	13.3 ± 0.47
						Fe3O4 (25 μ) + Rif (5 μ)	Bacillus cereus ATCC 13061	0
							Escherichia coli ATCC 43890	0
							Listeria monocytogenes ATCC 19115	0
							Staphylococcus aureus ATCC 49444	20.90 ± 0.50
							Salmonella typhimurium ATCC 43174	±0.34
α- Fe2O3	20	Spherical	Fe (NO3)3·9H2O, DW, & S. cordifolia extract.	GRS	NR	50	Escherichia coli	11.33 ± 0.58	NR	NR	NR	The ROS-mediated free radicals could disrupt the cell membrane via different interactions such as electrostatic, dipole, hydrogen, etc.	(121)
							Bacillus subtills	16 ± 1
							Staphylococcus aureus	13.67 ± 0.58
							Klebsiella pneumoniae	12 ± 1

^aNPs: Nanoparticles; Con.: Concentration; ZOI: zone of inhibition; ref: References; ROS: Reactive oxygen species; NR: Not reported; MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration; IC₅₀: Half maximal inhibitory concentration; FeCl₃.6H₂O: Ferric chloride hexahydrate; Fe (NO₃)₃·9H₂O: Ferric nitrate nonahydrate; DW: Deionized water; PEG: Poly(ethylene glycol); Kan: Kanamycin; Rif: Rifampicin; GRS: Green synthesis; GMAM: gray mangrove Avicennia marina; Amp: amphotericin; LPE: linear polyester; MH: Maize silky hairs; CCP: Leaves of Chinese cabbage; PMC: Psidium guajava-moringa composite; l-tyr: l-tyrosine; S. cordifolia: Sida cordifolia.

5.1.3. Other Chemical Synthesis Methods

IONPs can be synthesized using other chemically induced methods, including chemical precipitation, modified coprecipitation,¹²⁰ wet chemical, matrix-mediated method, hydrothermal, and laser ablation methods which are commercially available from several companies. In the modified coprecipitation method, cationic and anionic solutions are mixed for nucleation, and the aggregation of NPs is controlled by the pH and temperature.¹²⁰

The HDT produces IONPs with excellent crystalline structure at high temperature and pressure in an aqueous solution. The method also enhanced magnetism, uniform size distribution, and reduced aggregation. As a result, the IONPs produced through thermal decomposition would exhibit exceptional stability, while their compact size would significantly boost their antimicrobial effectiveness. For example, when Fe₃O₄ NPs are synthesized using HDT, they show more substantial antibacterial effects on Gram-positive than Gram-negative bacteria. IONPs displayed a ZOI range of 12–28 mm for Gram-positive and 12–22 mm for Gram-negative bacteria.⁶⁸ Therefore, IONPs synthesized through the HDT method demonstrated satisfactory antibacterial activity.

The liquid solution is subjected to pulsed laser beams to conduct the laser ablation synthesis procedure for synthesizing IONPs.¹²¹ The method consists of a high-power laser submerged in an aqueous solution, targeting the precursors. In this method, IONPs can be obtained using a single-step procedure. The IONPs produced using these methods are entirely surfactant-free, resulting in exceptional stability and purity. Thus, IONPs produced through the PLS method displayed moderate antibacterial activity against Gram-positive and Gram-negative bacteria.¹⁰³

5.1.4. GRS Method

GRS refers to a method where the biology-mediated synthesis of NPs is conducted. A range of biological elements, including microorganisms and leaf extracts like Chinese cabbage, mare silk, Psidium guajava,(122)Punica granatum, Zea mays,(123) and Sida cordifolia(91) could complete the synthesis procedure. This synthesis procedure is slightly modified,¹²¹ where the extraction of biological components is used as a reducing or stabilizing agent that is considered less toxic, convenient, economical, simple, eco-friendly, and biocompatible.^{30,71,99,124,125} IONPs synthesized through GRS acquire significant antibacterial activity against pathogenic bacteria compared to other chemical-induced synthesis methods^123,125 due to the inherent therapeutic characteristics of the plant extracts (Table 2).^85,126−128

According to a study by Singh et al.⁸⁵ the antimicrobial effectiveness of the MNPs depends on two parameters: (i) the precursors used to synthesize the nanomaterials and (ii) the size of the particles. The plant extracts utilized in synthesizing IONPs for antibacterial purposes consist of phytochemicals that possess preexisting therapeutic or medicinal capabilities. As a result, IONPs produced by GRS using the green precursors induce a combined antimicrobial action, which exhibits a comparatively heightened antibacterial activity than other chemically induced methods.^129,130Purpureocillium lilacinum was used to synthesize Fe₂O₃ and showed antibacterial activity against Gram-positive and negative bacteria, especially Gram-positive bacteria.¹³¹ This fungus is a well-known biocontrol agent used to produce leucinostatin antibiotics, which could be used against bacteria and fungus.¹³² Again, penicillium spp.,(133)Citrus leaf extract,¹³⁴Tamarix aphylla extract,¹³⁵Thymbra spicata leaf extract,¹³⁶Lawsone inermis extract,¹³⁷ and P. guajava leaf extract¹³⁸ possess prior antimicrobial activity. Hammad et al.¹³¹ reported that Fe₂O₃ NPs generated by Capparis zeylanica leaf extract showed approximately 23 and 22 mm ZOI for S. aureus and P. aeruginosa, respectively. Again, IONPs were synthesized using Penicillium spp and displayed an approximate ZOI of 12 and 11 mm for similar organisms.¹³⁹ In both cases, the IONPs generated by Capparis zeylanica showed a more pronounced antibacterial effect.

IONPs synthesized using Citrus leaf extracts inhibit the growth of B. cereus, K. pneumoniae, and B. subtilis. Similarly, Fe₂O₃ NPs produced from Tamarix aphylla and Thymbra spicata extracts are able to eradicate E. coli and S. aureus bacteria in a dose-dependent manner. Additionally, modifying the surface of IONPs with different types of antimicrobial agents, including kanamycin (Kan), rifampicin (Rif), and amphotericin (Amp), has proven to be more effective against microbes than the bare particles synthesized by GRS.^69,141,142 Moreover, IONPs synthesized via the GRS method have demonstrated notable antibacterial efficacy in compared to chemically synthesized methods (Tables 1, 2).

5.2. Size

IONPs have distinctive antimicrobial properties due to their physical and chemical characteristics, including the large surface area-to-volume ratio, superparamagnetic properties, and self-assembly.^147,148 These attributes are relevant to various factors, including their size, which influences their antimicrobial properties. Different synthesis procedures can influence the range of effective sizes of the IONPs.¹⁴⁹ Moreover, small IONPs exhibited enhanced stability and a higher surface-area-to-volume ratio, increasing free energy content, surface charge, and reactivity. The surface reacts with the microbes to achieve content stabilization, effectively diffusing into the cytoplasmic region and permeating the bacterial cell wall or membrane.^9,95,150 The heightened reactivity exhibited by NPs of smaller dimensions leads to an augmented generation of ROS, thereby promoting electron transfer due to the quantum confinement effect.¹⁵¹ Furthermore, to optimally utilize drug delivery and antibacterial activity, the NPs’ size should be less than 100 nm, commonly referred to as superparamagnetic iron oxide nanoparticles (SPIONs).^152−157

Generally, smaller IONPs show enhanced antimicrobial activity compared to larger IONPs. IONPs with a size of around 10–100 nm display moderate antibacterial activity against a range of bacteria. For instance, hematite with a size ranging from 13.13 to 24.93 nm showed excellent antibacterial activity, where the recorded ZOI for E. coli was 23 mm.¹³¹ Again, hematite with a slightly large size like 28.17 nm showed decreased bacteriostatic activity with a 19.5 mm ZOI.¹⁴⁰ The size difference between the two hematites was approximately 3–5 nm, influencing the antibacterial efficacy. Moreover, magnetite also showed significant antibacterial activity against numerous bacteria in sizes ranging from 8 to 260 nm. Moreover, where the size of IONPs is less than 10 nm and higher than 100 nm, the antibacterial activity of the IONPs decreases, such as IONPs ranging from 3.31 to 10 nm shows a ZOI of 11 ± 0.6 mm and 40 nm sized IONPs showed 21.5 ± 0.36 mm for K. pneumoniae.(69,139) Nevertheless, the antibacterial efficacy of this diverse range of IONPs lack to consistently correlate with their size due to other intrinsic parameters like precursors, synthesis methods, and coating materials (Figure 2). For instance, the effectiveness of antibacterial agents can vary depending on their dimensions and other inherent factors, like the materials used for their coating. In the case of both coated and uncoated IONPs, it was observed that their sizes varied within a range of approximately 6–254 nm and 3–200 nm, respectively, when employing the COP and GRS methods. However, it is often observed that smaller-sized IONPs tend to interact more frequently with pathogenic bacteria, resulting in dose reduction.^158−161 Consequently, smaller IONPs exhibit a greater level of antibacterial activity.^162−164

Figure 2.

An illustration of the numerous IONPs parameters that influenced the antibacterial activity. Here, AA: antimicrobial activity, AN: agar nutrient, AMH: agar Mueller-Hinton solution, LB: lysogenic broth, Amx: amoxicillin, TB: tryptic-soy broth, APD: agar potato dextrose solution, BN: broth of nutrient, BS: bacteriostatic effect, BC: bactericidal effect, and BG: bacterial growth.

5.3. Shape

The shape of the IONPs is crucial for their antibacterial activity. Numerous papers have documented different shapes of IONPs, such as hexagonal, quasi-hexagonal, and spherical. Interestingly, the majority of the research papers focused on the prevalence of spherical-shaped IONPs. These morphological differences are achieved by modifying the precursor molecules and temperature.¹⁶⁵ Diverse-shaped IONPs utilize different approaches to interact with bacteria; hexagonal IONPs are larger compared to spherical ones. As a result, the hexagonal IONPs interact with the bacterial cell wall due to their sharp edges, which results in cell wall distortion.^166,167 However, the spherical-shaped IONPs could diffuse into the intercellular space. Additionally, there is empirical evidence that displays this particular variant is not effective against all types of bacteria.^168−170 A study revealed that nanoparticles with a spherical shape exhibit the highest level of effectiveness in combating bacterial infections.¹⁷¹ Moreover, it is evident that spherical IONPs exhibit the most potent antibacterial activity. For example, when studying hexagonal IONPs using the COP method, the findings showed an average ZOI between 11 and 12 mm (Tables 1 & 2).⁵⁹ Conversely, IONPs with a spherical shape showed a 17 mm inhibition.⁹³ A larger ZOI suggests that spherical IONPs have superior antibacterial activity than their hexagonal counterparts.

5.4. Concentration

The concentration of IONPs significantly affects their antimicrobial action, depending on various aspects such as dose, saturation effect, and cellular absorption. The antimicrobial activity of IONPs frequently depends on their dose.^172,173 With the increased concentration, the bacterial cell surface may become saturated with IONPs, thereby increasing the probability of cellular uptake and consequently enhancing antimicrobial effects.^174,175 Nevertheless, saturation may impose a constraint on IONPs, thereby diminishing their antibacterial effectiveness.^176−179

The antimicrobial efficacy of the IONPs against pathogenic microorganisms is contingent upon their concentration. Due to the increase in concentration, a corresponding escalation in bacteriostatic activity was observed. Numerous studies have displayed the dose-dependent effect of IONPs against pathogenic microorganisms. For instance, Mehrabi et al.⁶⁸ have examined the dose-dependent effect of IONPs on several pathogenic bacteria, where the antimicrobial activity of IONPs was observed at different concentrations, at the lowest concentration of 25 μg/mL, approximately 20 mm and 15 mm, and at the highest concentration of 200 μg/mL, 28 mm and 22 mm ZOI for S. aureus and K. pneumonia. The elevated concentration would indicate a higher possibility of endocytosis and enhanced generation of ROS. The increased production of ROS would improve oxidative stress and quickly eliminate microbial cells. Another study displayed a concentration-dependent approach of Fe₂O₃ NPs against the bacteria B. subtilis, S. aureus, E. coli, and S. typhi (Table 1). At a 400 μg/mL concentration, the observed zone diameters were 11, 12, and 19 mm. However, at a higher concentration of 800 μg/mL, the observed zone diameters were 15, 16, 17, and 21 mm, respectively.¹²² The remarked zone diameter values exhibited a proportional relationship with the varying concentrations, whereby lower concentrations corresponded to smaller ZOI. In comparison, higher concentrations were associated with augmented zones of inhibition. Sharaf et al.⁸⁷ stated that the concentration of RHL-coated IONPs determines whether various bacterial strains exhibit bactericidal or bacteriostatic activity. The MIC and MBC concentrations of IONPs exhibited variation against distinct isolates of S. aureus, including MRSA, VRSA, and MSSA, which were 16, 32, and 64 μg/mL, respectively. Different isolates of E. coli bacteria also displayed multiple concentrations of MIC and MBC; hence, the increased concentration would elevate the antibacterial activity of the IONPs.

5.5. Precursors

IONPs can be synthesized using different iron salt precursors, e.g., chloride, sulfate, perchlorates, and nitrate salt precursors.¹⁸⁰ Moreover, the antimicrobial activity of IONPs can be significantly influenced by the utilization of distinct iron salt precursors during their synthesis. It is worth noting that the resulting IONPs exhibit varying levels of antimicrobial activity depending on the specific salts used during synthesis. The Fe₃O₄ sample, synthesized through the utilization of FeCl₃·6H₂O and FeCl₂·4H₂O, demonstrated a notable ZOI of 20 mm⁶⁸ while IONPs synthesized by Fe(NO₃)₃·9H₂O showed a 9 mm ZOI⁹⁹ for S. aureus. Furthermore, the iron precursor employed in the green synthesis process may also impact the bacteriostatic activity of the synthesized compounds. Fe₂O₃ NPs were synthesized utilizing various iron precursors via a sustainable and environmentally friendly approach known as green synthesis. Hematite was synthesized individually, employing FeCl₃·6H₂O and FeSO₄ as precursors, respectively. IONPs that were synthesized using FeCl₃·6H₂O exhibited a zone diameter of 21 and 23 mm¹⁴⁰ while IONPs synthesized by FeSO₄ exhibited a ZOI of 26, 19.5, and 17 mm for S. aureus, E. coli, and P. aeruginosa, respectively.¹³¹ The provided data (Table 1) indicated that IONPs produced using chloride salts exhibited greater efficacy compared to those synthesized using sulfate salt when tested against E. coli and P. aeruginosa. However, the IONPs synthesized using sulfate salt demonstrated superior effectiveness against S. aureus. Additionally, IONPs synthesized utilizing a chloride precursor exhibited a zone diameter measuring 26 and 21.5 mm, respectively.⁶⁹ On the contrary, Fe(NO₃)₃ precursor-derived IONPs showed 12 and 16 mm, ZOI for K. pneumoniae and B. subtilis, respectively.¹²¹ In this case, the chloride precursor was highly influential in inhibiting the microorganisms compared to the IONPs produced by nitrate salt. Hence, the inspection suggested that different precursors used to synthesize IONPs would influence their antimicrobial properties.^181−183

5.6. Surface Modification to Enhance Antibacterial Activity of IONPs

The surface modification of IONPs could influence their physical, chemical, and biological characteristics. There are various methods to modify the surface of IONPs; however, using coating techniques to modify the surface of IONPs is highly effective. Moreover, the application areas would vary according to the coating materials utilized on IONPs because the properties of the coating materials alter the activity of the IONPs. Several studies have been conducted which offer valuable insights into surface modification that increases the antibacterial activity of IONPs.^184−187 Hence, IONPs coated with antimicrobial agents such as gentamicin,⁹⁸ Kan, Rif, Amp,¹⁸⁸ poly(vinyl alcohol), teicoplanin,⁹⁴ dimethylformamide, sodium dodecyl sulfate,¹⁸⁹ and gallic acid (GA) form a nanocomplex, then these complexes tend to exhibit a combined effect against pathogenic bacteria (Tables 1 & 2). For instance, the bare IONPs exhibited ZOI, and the diameters were 17.33 ± 0.57 mm, 17.51 ± 0.57 mm, and 17.66 ± 0.57 mm while the IONPs conjugated with PEG+GEN demonstrated enhanced antimicrobial activity, with zone diameters of 23.66 ± 0.57 mm, 25.66 ± 0.57 mm, and 26.33 ± 0.57 mm against S. epidermidis, P. mirabilis, and A. baumannii, respectively. Hence, an increase in the diameter of the inhibitory zone by 7 mm was observed, which is attributed to the bacteriostatic properties exhibited by the coating material.⁹³ Additionally, similar results were observed as antibiotics, beta-cyclodextrin,¹⁰⁰ and l-tyrosine were coated on IONPs.¹⁴³

The coating material used to improve the antibacterial activity must have inherent bacteriostatic qualities; hence, utilizing various coating materials would influence the antibacterial activity of IONPs. For instance, Kan-coated IONPs showed an inhibition zone diameter of 10.35 ± 0.17 mm, 18.44 ± 0.41 mm, 16 ± 0.86 mm, and 13.17 ± 0.26 mm while Rif-coated IONPs exhibited 9.36 ± 0.28 mm, 9.62 ± 0.28 mm, and 21.99 ± 1.32 mm against B. cereus, E. coli, L. monocytogenes, S. aureus, and S. typhimurium, respectively.¹²³ Therefore, the different coating materials exhibited different ZOI. Additionally, utilizing multiple coating materials would display an increase in ZOI. For instance, the MICs of RHL-coated IONPs against various strains were found to be 4 μg/mL, 16 μg/mL, 32 μg/mL, 32 μg/mL, 64 μg/mL, and 64 μg/mL, while the MICs of RHL, PVA, GA, and p-CoA-coated IONPs against the same strains were determined to be 1 μg/mL, 4 μg/mL, 16 μg/mL, 16 μg/mL, 32 μg/mL, and 32 μg/mL E. coli (O26:H11), MSSA, E. coli (O78:H10), MRSA, E. coli (O157:H7), and VRSA, respectively. Hence, coating IONPs with multiple materials reduced the MIC dose to almost half against pathogenic bacteria compared to the MIC of single-coated IONPs. The coating materials RHL, PVA, GA, and p-CoA have significant antimicrobial properties as well as specific mechanisms to inhibit microbial cells.⁸⁷ As a result, after coating them with IONPs, the individual materials started exhibiting their bacteriostatic mechanisms and the antimicrobial activity of the whole nanocomplex increased. As these coated IONPs complexes have displayed enhanced antimicrobial activity compared to bare IONPs, it is conclusive to state that the surface modifications of the IONPs would enhance their antimicrobial activity.^190,191

Another approach could be doping impurities. It refers to the intentional introduction of specific foreign material into the lattice structure of the mother sample. Doping at various ratios to the other sample Fe₃O₄ could increase the antimicrobial activity. Since the dopant atom would influence the physicochemical properties of the IONPs, it would increase the interaction with the microorganism.¹⁹² Additionally, IONPs can achieve magnetic characteristics from dopant atoms, which could create severe vibration and damage the elasticity of the cell membrane and rupture the cells.^189,191,192 Therefore, doping would increase the antibacterial activity of IONPs.

6. Antimicrobial Mechanisms of IONPs

Studies have shown that IONPs have a moderate antimicrobial effect on various bacteria that are susceptible to it. In-depth research is being carried out to gain insights into the antibacterial mechanisms of IONPs. Until this point, these mechanisms involved the generation of ROS through the Fenton reaction leading to lipid peroxidation, DNA damage, and protein damage. Additionally, the presence of free radicals causes lipid peroxidation, which in turn initiates ferroptosis, resulting in oxidative stress and, ultimately, cell death.^193,194 Additionally, IONPs exhibit electrostatic interaction with bacteria and hinder cellular integrity. Thus far, these mentioned mechanistic understandings of IONPs have been theorized.

6.1. Cell Membrane Disruption

The cellular membrane of bacteria is composed of a bilayer of phospholipids interspersed with specialized proteins embedded within it. The semipermeable membrane exhibits hydrophilic polar heads and covalently linked hydrophobic nonpolar tails with ion channels.⁴⁹ Bacterial cell membranes are negatively charged due to highly electronegative groups on their constituent phospholipids and lipopolysaccharides.¹⁹⁵ IONPs can have either a positive or negative charge, leading to electrostatic interactions with the bacterial cell membrane. The antibacterial activity resulting from electrostatic interaction can differ depending on the surface charge of IONPs and the specific bacterial species. The surface charge of the IONPs interacts with the bacterial cell membrane through electrostatic attraction or repulsion and disrupts their integrity by increasing cell permeability, which allows frequent ion exchange into the cell and leads to cell death.¹⁹⁶ Additionally, IONPs adhere to the bacterial cell membrane, and due to their nanosized nature and high surface-to-volume ratio, they can cause physical disruption to the cell membrane.^30,149

Generally, synthesized IONPs consist of an overall negative charge due to the inherited hydroxyl group. However, the surface charge of the IONPs is dependent on the pH, being positive at low pH and negative at high pH. In both states, IONPs exhibit antibacterial activity through electrostatic attraction and repulsion. Positively charged iron ions present in IONPs have the potential to exert an influence on the integrity of cell membranes. An interruption occurs via electrostatic forces, upon the interaction of the iron ion with the cell wall. The metal ion then either attaches itself to the bacterial cell’s membranes or uses protein channels to enter the intracellular space.¹⁹⁷

6.2. ROS Production

One of the most auspicious antibacterial mechanisms exhibited by IONPs involves the generation of ROS through photocatalysis, cellular aerobic metabolism, and the Fenton reaction. ROS can be generated by reducing O₂ and the oxidation of H₂O.¹⁹⁸ In photocatalysis, UV light is required to generate ROS such as hydrogen peroxide (H₂O₂), hydroxy radical (⁻OH), and superoxide (·O₂^–).^199,200 However, ROS generated through the Fenton reaction do not require any solar light. The Fe³⁺ is reduced to Fe²⁺ by accepting an electron in the Fenton reaction. As Fe²⁺ is unstable in the presence of O₂, it is rapidly oxidized and produces ·O₂^–, reduces the Fe³⁺ to Fe²⁺, and their dismutation continuously produces H₂O₂. The reduced Fe²⁺ increases and reacts with H₂O₂, generating free radicals such as −OH.^201−204

The following reaction displays a generalized Fenton reaction for generating ROS.

These ROS inhibited bacterial growth in various ways, including peroxidation of lipids in the cell membrane, damaging DNA, inhibiting replication, transcription, and translation.^{196,205−211} Additionally, free radicals can reduce the antioxidant system, including enzymes and natural elements, e.g., catalase, peroxidase, ascorbic acid, and carotenes, which could deactivate enzymes and inhibit bacterial growth. Hence, ROS were held responsible for a cascade of malfunctions that resulted in bacterial mortality by inducing oxidative stress in the cell (Figure 3).

Figure 3.

A diagrammatic illustration of IONPs antibacterial and antibiofilm mechanisms. IONPs trigger oxidative stress and cell lysis by generating ROS, altering membrane depolarization, damaging protein or nucleic acid, inactivating enzymes, damaging mitochondria or ribosome, impairing efflux pump, disrupting cytoplasm and cell membrane. ROS: Reactive oxygen species; IONPs: Iron oxide nanoparticles.

6.3. Damage to Cellular Content

The cellular composition of a bacterial cell is primarily composed of DNA, proteins, and lipids. Once the IONPs have successfully traversed the cellular membrane, they possess the capability to engage with various constituents within the cytoplasmic milieu. Damage to DNA and proteins induced by reactive oxygen species (ROS) is commonly investigated in the field. However, it is worth noting that certain studies have suggested alternative outcomes based on the specific characteristics of ROS, such as their size, shape, and concentration.²¹² Although IONP’s toxicity is suggested, it also exhibits apparent genotoxic properties. The IONPs demonstrated that they directly intercalate with DNA, breaking DNA strands.^213,214 Additionally, the IONPs may interact with the proteins and enzymes necessary for ATP synthesis.²¹² These ions might obstruct ribosomal activities and stop them from synthesizing proteins. Gradually, interacting with the cytoplasmic content, replication, transcription, and translation might be inhibited by IONPs, which also have antibacterial properties.^23,196,215

6.4. Enzyme Inactivation

IONPs could inhibit bacterial growth by interfering with the enzymatic activities of the microbial cells. IONPs hinder the enzymatic pathways and inhibit bacterial growth by binding with the enzyme, denaturing the enzyme, interfering with the cofactors, and disrupting energy production.^216,217 IONPs can interact with either the surface or the active site of enzymes. Due to their nonspecific binding mechanism exhibited by IONPs, they possess the potential to function as irreversible enzyme inhibitors. IONPs exhibit diminutive dimensions, rendering them at the nanoscale level. Additionally, a molecular entity exhibits the inherent capability to act as an enzymatic inhibitor because it can imitate the native substrate and form a binding interaction with the enzyme’s catalytic site.²¹⁸

6.5. Efflux Pump

IONPs could inhibit bacterial growth by interacting with efflux pumps. The efflux pump is a transport system localized in the cytoplasmic membrane to expel harmful substances from the cell. So, bacteriostatic activity could be achieved by preventing the release of the efflux pump. There are possible mechanisms by which an efflux pump could be inhibited. The first possible mechanism to inhibit the efflux pump is the direct binding of IONPs to the efflux pump. The disruption of efflux kinetics could be another mechanism that inhibits microbial cell growth.²¹⁹ To comprehensively grasp the impact of the efflux pump on drug resistance, it is imperative to ascertain the kinetic constants. Hence, perturbing the efflux kinetics would exert an impact on the MDR efflux pump.²¹⁹ The initial mechanism elucidated the noteworthy potential of direct binding between IONPs and an efflux pump to impede the proliferation of microbial cells. Nevertheless, the interaction between IONPs and the efflux pump may give rise to certain challenges. One of the prevailing concerns pertains to the dimensions of the efflux pump and its inert response in the face of the IONPs.²²⁰ Therefore, IONPs might interact with the other membrane proteins to become associated with a nearby efflux pump.²²¹

7. Toxicity of IONPs

The utilization of IONPs as a therapeutic agent has become increasingly frequent in the clinical field. Therefore, conducting a cytotoxicity assay to assess the biocompatibility of IONPs both in vitro and in vivo is crucial. Frequently used techniques for investigating the toxicity and assessing the biocompatibility of IONPs in vitro include cell viability assay, LDH assay to examine cell membrane integrity, MTT assay to evaluate mitochondrial function, immunohistochemistry to detect markers of apoptosis, hemolysis assay, microscopic analysis of intracellular localization, and genotoxicity assessment for specific cell expression.^222−228 The toxicity of IONPs is influenced by various parameters, including their dimensions, morphology, surface coating, method of administration, attached peptide, medication, and targeting agents.^229−231 Smaller IONPs with a size lower than 10 nm are generally more hazardous than larger ones due to their higher surface area, which creates a higher possibility of penetrating the cells.²³² However, in the case of an in vivo system, smaller IONPs are rapidly eliminated through extravasation and renal clearance, whereas the spleen captures the larger ones through mechanical filtration.^233−235 Moreover, the surface charge can significantly influence the cytotoxicity of IONPs. The membrane potential of a cell is usually negative, and the surface charge of the IONPs is negative and sometimes positive; their interaction can be repulsive or attractive. The smaller NPs with a high surface area would have a higher change in ratio, increasing the electrostatic interaction between the cell and IONPs and damaging them. Furthermore, several coating materials are able to influence the toxicity of IONPs. For instance, longer exposure to IONPs could induce cytotoxicity due to the production of free radicals. However, toxic effects may be neutralized through PEGylation.²³⁶ PEG forms a hydrophilic barrier around IONPs, reducing the interaction between IONPs and cell membranes. More importantly, PEG coating enhances their antimicrobial activity and biocompatibility. Additionally, IONPs are less likely to cause significant health problems since Fe³⁺ is widely found in the human body. Hence, the release of the iron would not cause any major side effects in the in vivo system.^237−239

8. Limitations and Future Directions

IONPs have moderate antibacterial activity due to several factors, including their interaction mechanisms, nutrient deprivation, varying susceptibility, dose determination, and cytotoxicity.^240−242 IONPs interact with microbes through different mechanisms; however, these mechanisms depend on their size. Smaller IONPs have a larger surface area and can interact physically and more frequently with the bacterial cell membrane.²⁴³ On the contrary, the larger particles are not able to interact much frequently. Due to having a smaller surface area to volume ratio, their determined dose to eradicate bacteria increases.^244,245 Moreover, dose determination becomes extremely difficult when the pathogens have variable susceptibility. Furthermore, IONPs are able to interfere with microbial nutrient uptake, as most pathogenic bacteria require iron for their growth and survival.^246,247 Additionally, a few of the IONPs interact with cells, producing ROS that can be toxic for cells and induce pro-inflammatory mediators.^248−251

Though IONPs have several limitations as therapeutic agents, these limitations can be minimized using biocompatible precursors and surface modification. For instance, in the GRS synthesis method, bioreductants, which consist of phytochemicals with prior antimicrobial activity, are used instead of chemicals.^252,253 Therefore, a combined mechanism is initiated against eliminating bacteria; hence, the potency of IONPs increases.²⁵⁴ Additionally, surface modification approaches can help address IONPs’ main drawbacks, such as stability issues, cytotoxicity, biocompatibility, and moderate antibacterial activity.^149,255 Biopolymers such as PEG and chitosan are used as coating material to modify the surface of IONPs.^256,257 These biopolymers consist of antibacterial and biocompatible properties.²⁵⁸ Hence, surface-modified IONPs would be more potent as an antimicrobial agent because these biopolymers would compress the size of the particles and increase the surface area to volume ratio, resulting in a combined mechanism against pathogenic bacteria and acquiring increased bioavailability while keeping the cytotoxicity to a minimum by reducing the interaction between IONPs and tissues.^259−261

IONPs exhibit moderate antibacterial activity as a therapeutic agent against pathogenic bacteria since the dose of IONPs would vary depending on the bacterial species, size of IONPs, and ROS production capabilities. For better and more precise outcomes, the GRS synthesis method and surface modification can be utilized to produce stable IONPs with enhanced biocompatibility and antibacterial activity. Furthermore, IONPs can be used in different clinical sectors, including cancer detection and treatment, magnetic hyperthermia, multimodal imaging, soil and water purifications, and biosensors.^{42,196,262,263}

9. Conclusion

The antibacterial activity of the bare and coated IONPs depends on the synthesis methods, size, concentration, precursors, surface charge, and the materials used for coating. Among several synthesis methods, IONPs synthesized through GRS exhibited notable antibacterial efficacy. The phytochemicals with prior antimicrobial properties used in producing IONPs displayed a combined mechanism against bacteria. Additionally, IONPs expressed moderate antibacterial activity within the size range of approximately 10–100 nm. They exhibited higher cellular uptake and efficient antibacterial activity within this range, while IONPs with sizes smaller than 10 nm were less effective against bacteria. Furthermore, their properties to inhibit bacteria depend on their diminutive dimensions, and spherical-shaped IONPs show more significant potential for inhibiting bacteria than other shapes. Moreover, depending on the utilization of different iron salt precursors (e.g., sulfate, chloride, and nitrate) the antibacterial properties of IONPs vary. Among all the iron salt precursors, chloride salt has been proven to be the most effective precursor utilized to synthesize IONPs as an antibacterial agent. Although IONPs are used as therapeutic agents, they show moderate antibacterial activity. The moderate activities are increased by using different biopolymers (e.g., PEG, chitosan, L-try, antibiotics) as coating material. Surface modification via biocompatible coating material or drug candidates would increase stability, enhance biocompatibility, and simultaneously augment their antibacterial efficacy to address the pressing issue of infectious diseases caused by AMR organisms.

Author Contributions

Conceptualization, M.S.S.; writing original draft preparation, K.N.S., S.U., M.S.S., figure drawing S.U., M.H.R.; review and editing, S.U, M.H.R., and M.S.S.; project administration, M.S.S., M.H.R., and S.U.

The authors declare no competing financial interest.