The synthesis procedures physical, chemical, microbial that are often employed in their fabrication are also outlined. The interaction of various nanoparticles with microbes is described with attention given to the role of additives in the form of solvents, surfactants, capping materials. Commonly used experimental and analytical techniques that are often used to evaluate and determine the toxicity of nanoparticles towards different microorganisms are presented and comparative assessments on the differences between these procedures are described.
The brief ends by explaining the toxicity of metal and metal oxide nanoparticles to microorganisms. About the Author Dr. Anil K. Suresh, Ph. Pharmaceutical Analysis E-Book. David G. Joseph C. Iwona Wawer. Diamondoid Molecules. G Ali Mansoori. Wolfgang Pompe. Nonionic Surfactants. Vaughn Nace. Microfluidic Devices in Nanotechnology. Challa S. Essentials of Pharmaceutical Preformulation. Simon Gaisford. Applied Physical Pharmacy, Third Edition. Mansoor Amiji. Understanding Bioanalytical Chemistry. Victor A. Engineered Nanoparticles. Ashok K Singh. Data-Handling in Biomedical Science.
Peter White. OAT Prep Plus Drug Delivery. Mark Saltzman. Nanomedicine - Overview of a new science. Marco Casella. Advances in Photocatalytic Disinfection. Taicheng An. Debasis Bagchi. Malgorzata Baranska. Progressive Management. Membrane Processes in Biotechnology and Pharmaceutics. Catherine Charcosset. Supramolecular Chemistry. Peter J. Soshu Kirihara.
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Gianni Ciofani. Physical Properties of Tissues. Francis A Duck. Thomas J Webster. Patrick F. Nanotechnology to Aid Chemical and Biological Defense. Terri A. Plasma Medicine. Alexander Fridman. Physiological Models in Microbiology. Modeling of Microscale Transport in Biological Processes. Finally, the Ag-BPEI-NPs induced a response similar to any cationic particle signifying that bactericidal activity is the most important contributor to the charge [ 30 ]. During NP fabrication, a capping agent is added to increase the stability and facilitate the dispersion of the NPs.
These agents may have a direct effect on the toxicity of the NPs, likely due to their ability to reduce NP agglomeration [ 6 , 7 , 12 , 45 , 46 ]. The capping agent chitosan has been shown to possess antibacterial activities against E. It should be stressed that the experiments were performed in P. As a result of the toxicity generated by the chemical compounds used for NP fabrication, green technologies were developed to overcome this issue.
The presence of reducing compounds in plant extracts have led to their increased usage over the last few years. Furthermore, functional groups can be added to the surface of the NPs. For example, the morphology of Ag-NPs changes depending on the stabilizer used [ 45 ]. Using a UV—Vis absorption peak, it was discovered that increasing the concentration of plant extract leads to a stronger binding of the capping agents and the biomolecules. Ultimately, the study concluded that the positively charged detergent cetyl trimethylammonium bromide CTAB enhances NP toxicity by directing the adsorption on specific crystal planes of the NPs.
Moreover, an aggregation process that occurs between the negatively charged cell wall and the presence of CTAB has been proposed, suggesting a synergistic effect between the CTAB and NPs [ 39 ]. Treating NPs with halogens can increase their antibacterial activity [ 41 ]. For instance, a formulation of NPs using an aerogel was prepared with MgO and Cl 2 or Br 2 to solve the problem of the high toxicity and vapor pressure associated with halogens [ 41 ]. The aerogel formation meant that Cl 2 was converted into a dry powder form with no loss of activity.
The resulting NPs were equally active against both Gram-negative and -positive bacteria and even had slight activity against endospores. Authors concluded that the high activity was likely due to the abrasiveness, high surface area, and oxidizing power of the halogen [ 41 ]. NPs are constantly undergoing dissolution because of the electrochemical potential in solution. It has been shown that the antibacterial activity of NPs is based on and proportional to the release of ions, although other mechanisms can be involved as well [ 30 , 38 , 40 , 48 , 49 , 50 ].
The concentration of NPs directly effects toxicity because a larger concentration of NPs releases more ions [ 51 , 52 ] with a concomitant increase over time [ 53 ], correlating with findings that longer incubation time decrease viability. It has been found that E. Using inductively coupled plasma mass spectrometry, it was found that Al 2 O 3 contained 0. Ions are often responsible for toxicity. When metal ions in solution are exposed to bacterial cell is, they become uniformly distributed in the environment surrounding the bacterial cell with no specific localization.
In contrast, NPs that interact with the bacterial cell wall produce a focal source of ions continuously release ions, and causing more toxicity to the cells [ 48 ]. The large generated ion concentration further helps to penetrate the cells. As a consequence, the NP dissolution is localized around the bacterial cell membrane, with the kinetic of dissolution depending on the size and shape of the NP. The surface morphology of the NPs have a profound effect on the activity of the NPs and when the surface of the NPs are rougher, the dissolution occurs faster [ 50 ].
Additionally, the larger surface area to volume ratio in smaller NPs results in faster dissolution. NPs have higher antibacterial activity than their bulk counterparts [ 12 , 51 , 52 , 53 , 54 , 55 ]. While antibacterial activity is evident from ions alone, the fact that NPs are more toxic indicates that other mechanisms contribute to toxicity.
However, contradictory evidence has been reported. The release of ions from NPs appears to be element dependent. To attain the same toxicity level as a fixed concentration of Cu-NPs would thus require an increased amount of Ag-NPs is necessary to attain the same toxicity level as a fixed concentration of Cu-NPs, consistent with the idea that ion release is crucial for antibacterial activity. The fact that Ag-NPs are still more efficient to kill bacteria than Cu-NPs regardless the ion generation , can be explained by the essentiality of Cu in physiological systems.
Cu is an essential element playing a role as a co-factor for different enzymatic systems, such as those involved in redox reactions essential to cellular respiration cytochrome oxidase and superoxide dismutase antioxidant defense [ 57 ]. These proteins possess an unusual number of cysteine residues in their sequence and probably have a role in toxicity defense against metals [ 61 ].
Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations
Other Cu-binding proteins are the CueO multi-Cu oxidase [ 63 ] and the CusCFBA multicomponent efflux transport system [ 64 ], both contributing to the intracellular homeostasis of Cu and protection of the bacterial cell. Microbes have developed many systems to neutralize antibiotics. We describe, as an example, a few of the mechanisms of resistance to antibiotics in bacteria, which may potentially be relevant to NP resistance Fig. The hydrophilic nature of common antibiotics like beta-lactams and aminoglycoside makes cell penetration difficult.
NPs represent an attractive solution for the hydrophilicity barrier because they can often penetrate cells, especially in phagocytic cells macrophages , which may engulf NPs and increase their intracellular activity [ 44 ]. Mechanisms of selected antibiotic classes and antibacterial resistance. Aminoglycoside antibiotics diffuse through porin channels of Gram-negative bacteria and are then actively transported into the cell where they irreversibly bind to the 30S ribosomal subunit, inhibiting protein synthesis [ 65 ].
On the other hand, beta-lactam antibiotics attach to penicillin-binding proteins and ultimately inhibit cell wall peptidoglycan synthesis and inactivate autolytic enzyme inhibitors [ 65 ]. Because this class of antibiotic facilitates a breakdown of the cell wall, it is possible that NPs are more effective combined with antibiotics simply because it is easier for the NPs to enter the cell. The reverse is true as well, when NPs disintegrate the cell wall, it is easier for antibiotics to enter the cell, especially aminoglycosides whose mechanism of action does not involve cell wall breakdown.
Both aminoglycosides and beta-lactam antibiotics contain hydroxyl and amino groups that could interact as targets of the NPs [ 65 ]. It is worth noting that NPs have not been show to undergo a morphological change with the addition of antibiotics [ 44 ]. Antibiotic-conjugated NPs exhibit a higher antibacterial activity than the antibiotic alone or NP alone, indicating a synergistic effect and hinting that NPs and antibiotics use different antibacterial mechanisms [ 44 , 65 , 66 ].
In addition, a study using E. However, an unintentional binding may still have occurred between NP and antibiotic [ 65 ]. The exposure of NPs to bacterial cells can lead to membrane damage caused by NP adsorption sometimes followed by penetration into the cell [ 16 , 36 , 41 , 48 ]. Many studies suggest that adsorption on the cell wall following its disintegration is the primary mechanism of toxicity [ 13 , 36 , 48 , 68 ]. Adsorption of NPs leads to cell wall depolarization, which changes the typically negative charge of the wall to become more permeable.
It has been reported that the bacterial cell wall become blurry, indicating cell wall degradation as shown by a laser scanning confocal microscope [ 5 ]. In this study, authors suggested a bimodal mechanism of action of Ag-NPs. In the first step, the cell wall is destroyed with subsequent penetration of NPs. Since the production of ROS has been shown to counteract the cell built-in antioxidant defense and lead to cell wall into the cell damage, it is possible that the production of ROS plays a part in the primary step as well [ 69 ].
Ag-NPs themselves have also been found to associate with the cell wall [ 48 , 54 ]. This is hypothesized to be a source of toxicity as this association can result in degradation, allowing ions to enter into the cytosol. Ag-NPs also have an ability to cause irregular pit formations on the cell wall [ 39 , 47 ], which facilitate ions entering the cell and halts transport regulation as observed by transmission electron microscopy.
Criticism has been raised regarding the current bacterial cell analysis methods due to the common assumption that the cell surface is uniform with all embedded molecules having a totipotent binding affinity, as well as the assumption that all cells in a population have the same surface tension [ 71 ]. This assumption was supported by challenging the assumption of uniformity by binding Au-NPs to S. It was found that carboxylic acid functionalized NPs exhibited a preferential attachment to the subpolar area of the cell.
When a mutant lacking type IV pili proteins was substituted, there was no longer a binding preference [ 71 ]. Contrary to many findings of cell permeation, the interaction of MgO-NPs with the cell wall is the main source of toxicity to bacteria even though no cell penetration occurs [ 68 ]. Similarly, Mg OH 2 -NPs electrostatically adsorb onto the bacterial cell wall and destroy the cell wall with no NP penetration into the cell, but NP aggregation has been observed on the cell surface [ 43 ]. Similar studies have reported that when NPs interacts with the bacterial cell wall, penetration does not always occur [ 16 , 28 ].
Even when toxic NPs adsorb onto the surface and enter the periplasmic space, internalization is not always toxic [ 13 ], which signifies that aggregation may constitute a significant source of toxicity. Extracellular NP aggregation has been observed in numerous studies, sometimes with NPs aggregating together and sometimes with NPs aggregating with bacterial cells [ 12 , 13 , 16 , 36 , 41 , 47 , 52 , 72 ]. The aggregation can lead to cell envelope damage and changes in the cell of smoothness and thickness [ 41 ].
However, it has been reported that capping ZnO-NPs with thiol prevented clumping, suggesting that capping is a potential solution for the aggregation issues [ 12 ]. NP aggregation can also be a serious problem because if the NPs are aggregating with one another, interaction with the bacterial cell wall is prevented, inhibiting toxic activity [ 13 ]. NP aggregation can be predicted from the measurement of zeta potential, which indicates the stability of colloidal suspensions [ 73 ].
Even at the optimal zeta potential, NPs can still aggregate with each other as a result of protein complexation. In this regard, the thermodynamics of protein-NP complexation was investigated [ 74 ] using different sizes of Au-NPs and proteins, such as green fluorescent protein a beta barrel protein [ 75 ], BSA a triangular prismatic protein [ 76 ], and PhosA an orthorhombic shaped protein [ 77 ]. The study reported that proteins bind to the Au-NPs in different ratios.
Consequently, a complex formation between protein and NPs is independent of the aggregation induced by the zeta potential and may govern the aggregation of NPs on the cell wall of the bacteria. When the bacterial strains E. The damaged cells were viewed using transmission electron microscopy imaging and it was revealed that the cell wall had physically separated from the internal cellular environment and that electron dense aggregation of compounds were surrounding the lysed cell.
Co-Relating Metallic Nanoparticle Characteristics and Bacterial Toxicity | ecejyredagij.ml
Ag-NPs are able to create a barrier between the cell wall and the cytoplasm more effectively in Gram-negative E. Further studies treated E. This phenomenon is known to happen during plasmolysis, the process of a cell losing water and it has been hypothesized that this may occur due to cell wall destabilization causing a release of ions internally [ 52 ]. Ag-NP exposure was found to reduce cell membrane integrity with an increase in the permeability, likely due to the neutralization of the cell membrane surface charge.
When E. Microscopic imaging showed that treated cells had disrupted membranes with intracellular components pooling around the cells due to membrane leakage. Similar results of lost cell integrity and appearance of cellular debris outside of the cell were observed when P. Interestingly, an elongation of the cells was also observed, possibly due to stress conditions arresting cell division.
Moreover, E. ROS are species of oxygen that are highly reactive and are produced during basic metabolism. Universal intracellular mechanisms of defense have evolved to cope with this undesired chemical to avoid damage to essential biomolecules in the cell. However, under high levels of stress, the levels of ROS can increase significantly and it is hypothesized that their generation is one of the focal NP mechanisms of action that inhibit bacterial growth [ 12 , 30 , 68 , 72 , 80 ]. ROS are produced when oxygen enters undesired reduction states and transforms into free radicals, superoxides, and peroxides, rather than water.
A stress on the cell, such as UV light, DNA damage, and NPs, can cause ROS production to increase to a level that is toxic to the cell [ 81 ], and can cause cell damage or cell death [ 81 ]. NPs have been shown to generate free radicals with an increase in NP concentration leading to a concomitant increase of ROS [ 29 , 34 , 50 , 69 , 82 ]. Even C. The increasing levels of NPs in the environment may cause a perturbation to the native bacterial populations, such as nitrifying bacteria with essential roles in the transformation of ammonia to nitrates in municipal sewage treatment.
When nitrogen-cycling bacteria are exposed to sublethal concentrations of Ag-NPs nitrifying genes are upregulated, however, upon exposure to higher concentrations of Ag-NPs, the upregulation stimulus is no longer present [ 83 ]. It is possible that at high concentrations of NPs, loss of cellular integrity interferes with the generation of ROS. The oxidation state of the metal in the NPs may contribute to the bactericidal effect.
Both intracellular and extracellular ROS are able to disrupt cell membranes [ 38 ]. One way of alteration of the cell membrane is by lipid oxidation which can easily be generated by free radicals [ 50 ]. Interestingly, in the case of S. Some ROS such as OH radicals are negatively charged, meaning that they cannot easily penetrate the negative charged cell membrane [ 12 ], regardless of Gram classification.
However, H 2 O 2 is a commonly produced ROS which is able to penetrate the cell membrane and kill bacteria [ 12 ]. ROS formation at the cell wall is due to positive NPs interacting with the negative charge on the cell wall [ 30 ]. Damage is further increased by the production of ROS, which has been shown to counteract the antioxidant defense built into the cell by surpassing its capacity, damaging the cell membrane [ 68 ].
Some studies found that free radicals are able to induce cellular membrane damage [ 50 ] and the oxidative stress can lead to lipid peroxidation, inhibiting bacterial growth [ 71 , 81 ].
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Surprisingly, ROS are able to damage cellular DNA without visible membrane damage, suggesting a complex mechanism of toxicity [ 29 ]. It is not clear yet whether oxidative stress is the primary or secondary mechanism of killing. H 2 O 2 specifically largely contributed to the antibacterial activity [ 38 ], suggesting that catalytic oxidation is the main mechanism in the bactericidal process. However, another study found that oxidative stress is a secondary mechanism in the bacterial killing process [ 83 ]. Oxidative stress can lead to and increased depletion of GSH [ 71 , 81 ].
This reaction protects the cell from harmful redox reactions by scavenging ROS molecules [ 69 ]. It is hypothesized that NP concentration decreases as the NPs interact and bind with organic materials in the culture broth and damaged cell components [ 30 ]. Media components that may interfere include sodium citrate, phosphates that form Zn 3 PO 4 2 , amino acid, and peptides [ 84 ].
Moreover, bacteria treated with CuO- and Ag-NP showed that bacterial secretion of exopolysaccharides interacted with the NPs, extracellularly trapping the NPs and decreasing toxicity [ 50 , 84 ]. Once inside the cell, the NPs release ions, which target multiple sites simultaneously. Ag-NPs are commonly used to investigate protein-binding properties due to their affinity for thiol groups [ 5 , 47 ].
Amongst the enzymes with a similar high affinity for Ag-NPs are tryptophanase, alcohol dehydrogenase, and cytochrome C, as demonstrated in a time-dependent reaction, suggesting a hierarchical binding to proteins [ 85 ]. The non-enzymatic proteins that Ag-NPs bind to are involved in membrane integrity, such as membrane porins OmpA and OmpB , chaperonins, and periplasmic peptide binding proteins [ 85 ]. The high affinity of the periplasmic peptide binding protein towards Ag-NPs may explain why these NPs accumulate in the periplasmic area of the bacteria [ 29 ].
Thiol is the functional group on the amino acid cysteine. Cysteine is very important in biological reactions due to disulfide bridging which is crucial for proper protein folding and function, as well as, its nucleophilic role in catalytic reactions. In the specific case of cell wall synthesis enzymes, it has been reported that the protein-NP interaction occurs in the SH group of the mannose phosphate isomerase, leading to an interruption of cell wall synthesis with a concomitant leaching of internal components, and cell death [ 5 ].
NPs exposed to bacterial cells have been shown to cause changes in the genomic and proteomic profiles, suggesting that the presence of NPs primes an adaptation of the cells to the new NP-containing environment. Another study reported that E.
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Most other changes in expression levels seemed to indicate that Ce disrupts respiration or iron homeostasis because many iron uptake genes responded to NP treatment [ 16 ]. Escherichia coli treated with MgO-NPs differentially regulated proteins with 83 being downregulated [ 68 ]. These proteins were mostly part of central metabolism, genetic transcription, and others needed for cellular function.
The upregulated genes were thiamine-binding periplasmic protein and proteins associated with riboflavin metabolism, suggesting that the upregulated genes did not seem to bear relevance to the toxicity of MgO-NP exposure. The increasing ROS level in a bacterial cell will induce the transcription of genes involved in the cellular protection against ROS. In contrast, not all the NPs are able to elicit an antioxidant response as in the case of E.
Pseudomonas sp. Both TSA and AhpC belong to an antioxidant family of enzymes called peroxiredoxins, which protect the cell from peroxide damage and are expressed during an oxidative stress [ 89 ]. This upregulation supports the hypothesis that Ag-NPs induce oxidative stress in cells because of the increasing level of these enzymes produced to cope with the increasing ROS levels. KHGA is associated with sugar metabolism, converting sugar acids, hexonates, and hexuronates into pyruvate and glyceraldehydephosphate [ 90 ].
It also regulates glyoxylate levels and prevents toxin accumulation [ 91 ]. Translational ribosomal proteins S2 and L9 are involved in translational regulation and also have functions in structure and stress regulation [ 92 ]. In the case of E. Both ahpC upregulated by Ag-NPs and aphF are involved in peroxide metabolism, but are differentially regulated upon exposure to different NPs. This suggests that different pathways for upregulation are involved [ 88 ]. A similar observation of ahpC downregulation was reported in another study when E. Another gene triggered by high peroxide levels is katE , a catalase that decompose H 2 O 2 to protect the cell from ROS damage.
When the gene katE is absent gene knock out , an Ag-sensitive phenotype is induced [ 30 ]. This gene regulates redox reactions and is involved in peroxide metabolism and protection [ 87 ]. Other genes involved in these processes that were found to be upregulated are sodA , sodB , sodC , and katG. This could be due to a feedback loop of the protein regulating the gene or it could be due to progressive cell membrane disintegration, leading to the entire cell no longer being able to regulate gene expression. For instance, E.
The 30S subunit is responsible for proper base pairing between the codons and anticodons. As a result of its denaturation, the expression of other proteins are suppressed, such as succinyl-CoA synthetase which is necessary for catalysis of intracellular ATP production [ 93 ]. The deficiency in necessary proteins and enzymes to run the citric acid cycle leads to a deficiency in ATP.
This could explain the ATP depletion observed upon exposing E.
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Bacteria are exposed to stress originating by multiple sources in the environment. To adapt and survive the stress, bacteria respond by activating and coordinating a complex network of genes that cope with the external stimulus for an effective response. Two of the most important stress responses include the upregulation of envelope stress and heat shock proteins. Both have been observed when bacterial cells have been treated with NPs. It has been found that the expression of cell envelope proteins seems to be upregulated upon Ag-NP exposure.
This was detectable because the proteins remained in a precursor form due to the Ag-NP inhibition of the process of conversion into shorter, mature forms in E.