biomimetics
Review
Metal Oxide Nanoparticles as Biomedical Materials
Maria P. Nikolova 1, *
1
2
3
*
and Murthy S. Chavali 2,3
Department of Material Science and Technology, University of Ruse “A. Kanchev”, 8 Studentska Str.,
7017 Ruse, Bulgaria
Department of Chemistry (PG Studies), Shree Velagapudi Ramakrishna Memorial College, Nagaram Guntur
District, Andhra Pradesh 522 268, India; ChavaliM@gmail.com
Nano Technology Research Centre, MC Education, Training, Research and Consutancy, Tenali, Guntur
District, Andhra Pradesh 522 201, India
Correspondence: mpnikolova@uni-ruse.bg; Tel.: +35-9888785133
Received: 27 April 2020; Accepted: 1 June 2020; Published: 8 June 2020
Abstract: The development of new nanomaterials with high biomedical performance and low toxicity
is essential to obtain more efficient therapy and precise diagnostic tools and devices. Recently, scientists
often face issues of balancing between positive therapeutic effects of metal oxide nanoparticles and
their toxic side effects. In this review, considering metal oxide nanoparticles as important technological
and biomedical materials, the authors provide a comprehensive review of researches on metal oxide
nanoparticles, their nanoscale physicochemical properties, defining specific applications in the various
fields of nanomedicine. Authors discuss the recent development of metal oxide nanoparticles that were
employed as biomedical materials in tissue therapy, immunotherapy, diagnosis, dentistry, regenerative
medicine, wound healing and biosensing platforms. Besides, their antimicrobial, antifungal, antiviral
properties along with biotoxicology were debated in detail. The significant breakthroughs in the field
of nanobiomedicine have emerged in areas and numbers predicting tremendous application potential
and enormous market value for metal oxide nanoparticles.
Keywords: metal oxides; nanotoxicity; oxidative stress; ROS; magnetic nanoparticles
1. Introduction
Progress in nanotechnology and interdisciplinary research enables the production of nanosized
materials with unique physical and chemical properties that make them suitable candidates for biomedical
applications. Generally, nanotechnology includes synthesis and control of matter at dimensions of
a few hundred nanometers that enable specific size-dependent properties [1]. Nanoparticles (NPs)
dedicated to nanomedical applications ought to have a preferential size of less than 200 nm [2]. Due to
their small size and large surface area, NPs show enhanced colloidal stability and, therefore, increased
bioavailability demonstrating the ability to cross the blood-brain barrier, enter the pulmonary system and
adsorb through endothelial cells [3]. Specifically, metal oxide NPs (MONPs) possess some advantages
such as high stability, simple preparation processes, easy engineering to the desired size, shape and
porosity, no swelling variations, easy incorporation into hydrophobic and hydrophilic systems and
easy functionalization by various molecules due to the negative charge of the surface, that make them
a promising tool for biomedical applications [4]. Since MONPs react with in vivo systems differently,
depending on their size, shape, purity, stability and surface properties, it is necessary to characterize their
morphology. Based on the number of dimensions, which are not confined to the nano-range, MONPs can
be classified into zero-, one-, two- and three-dimensional [5] as represented in Figure 1. Zero-dimensional
nanomaterials include NPs, nanoclusters, quantum dots and so forth that have all dimensions on the
nanometer scale. For the one-dimensional NPs such as nanorods, nanotubes, nanowires, nanofibers,
one dimension is outside the nanoscale. Two-dimensional or planar nanomaterials like nanosheets,
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‐
nanocoatings, nanofilms,
nanoplatelets, have two dimensions outside the nanoscale. Dendrimers,
bundles of nanowires or nanotubes, nanopillars, nanoflowers, multi-nanolayers and so forth belong to
‐
‐
the group of three-dimensional NPs. They are formed when nanomaterials aggregate in a certain size
bigger than 100 nm in all three orthogonal directions. For the synthesis of these MONPs, various methods
have been introduced that are summarized in previous reviews [6]. Generally, MONPs have highly ionic
nature and can be organized with crystal morphologies exhibiting various reactive sites and corners.
The deposition of MONPs on the desired position with a nanoscale resolution on a certain substrate
has a promising potential to realize nanodevices for various applications in chemistry, electronics,
optics and biomedicine [7].
‐ one-,
‐ two‐ and three-dimensional
‐
Figure 1. Scheme of zero-,
nanostructured materials with different
‐ 1-D,
‐ 2-D,‐ and 3-D indicate
‐
‐ two-‐ and three-dimensional
‐
‐
morphologies. 0-D,
zero-, one-,
NPs, respectively.
The biomedical application of NPs for diagnostics and therapeutics (drug delivery and enhanced
performance of medical devices) rapidly progresses during recent years. The usage of MONPs
for diagnostics and therapy offers many advantages of modern medicine. The engineering of
‐
water-dispersible NPs allows these particles to be used in countless basic or applied biomedical
researches. Right now, NPs are used in diagnostic for imaging of plentiful molecular markers,
of genetic and autoimmune diseases, malignant tumors, photosensitizers in photodynamic therapy
and target delivery of drugs [8]. Today NPs for biomedical purposes are adopted as diagnostic
imaging materials such as viable fluorescent-labeled particles (semiconductor quantum dots), magnetic
‐
resonance imaging (iron nanooxides) and so forth. This work was aimed at exploring the complications
to induce further investigations by summarizing new approaches, benefitting the features of MONPs
and successful studies of applications of nano metal oxides in nanomedicine. A special interest has
been shown towards green synthesized nano-oxides and their antibacterial
activity as well as towards
‐
the toxic effects of MONPs on the organism.
2. Physical-Chemical
Properties of Nano-Oxides
‐
‐
When entered into the body, NPs interact with biofluids and cell biomolecules that facilitate the
physical transfer of the particles into the inner cellular structures [9]. As the biological response to NPs
depends on the variety of factors such as size, morphology, aggregation and so forth, the controlled
synthesis techniques are focused on attaining of NPs with tailored morphological configurations,
sizes, distribution and stability. The main characteristics influencing the performance of NPs when
contacting with cell can be summarized as follow:
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2.1. Shape and Size
The size determines the surface-to-volume ratio and can affect the biodistribution and material
uptake [10]. Since the openings of the blood vessels in many tumors are less than 200 nm and the
mammalian vasculature has a pore size of about 5 nm and below this size, Elsabahy and Wooley
suggest that the intermediate size (20–100 nm) of NPs has the highest potential for in vivo application
due to the ability to propose enough circulation time [11]. The hydrodynamic size is one of the most
important factors in determining the distribution and clearance of NPs in the organism. Research
reports suggested that iron oxide NPs >100 nm in diameter were rapidly trapped in the liver and
spleen through macrophage phagocytosis whereas those with the size of <10 nm were eliminated
through renal clearance [12]. Numerous experiments pointed out that smaller than 5 nm NPs overcome
cell barriers via translocation or other nonspecific mechanisms while larger particles entered the cell by
pinocytosis, phagocytosis or other specific or non-specific cell transport mechanisms [8] Regarding
the shape, nanosized oxides with different morphologies such as nanorods, nanospheres, nanocubes,
nanowires, nanotubes and so forth have been synthesized. In vitro evaluation on iron oxide nanorods
and nanospheres carried out in human carcinoma cells indicated that hematite nanorods were more
quickly and in a higher extend internalized than nanospheres [13]. The more favorable cellular uptake
of rod-shaped NPs, as opposed to spherical NPs, Andrade et al. [14], explained with the larger area of
contact between the cell membrane and rod-shaped NP. Comparing tripod with spherical iron oxide
NPs, it has been found that tripod particles had lower cytotoxicity in HeLa and Hepa 1-6 cells at
concentrations between 0.022 and 0.35 mg Fe/mL [15]. Other authors showed that the internalization
of nanoflowers was higher than single-core NPs [16]. It follows that the effect of the shape on NP
toxicity is relevant to high aspect ratio conditions. The different combinations of shapes and sizes with
specific properties of nanomaterials have to be also considered.
2.2. Surface Area and Surface Energy
The increased surface area and energy of NPs result in a drastic reduction of thermodynamic
stability hindering the degree of uniformity, shape and size [17]. The surface of crystal oxide NPs
consists of oxygen atoms with a lower coordination number due to the disruption of the crystal
periodicity that leads to violation of the electro-neutrality between anions and cations and, therefore,
surface change [18]. As NPs become smaller the percentage of surface located atoms increases
concerning the total amount of atoms. The numerous edges and corners of the particles are potential
reactive surface sites. The high surface-to-volume ratio provides NPs with a large number of active
sites, small size and particular shape that could control reactivity. The particles with a larger surface
area have a higher percentage of interaction as compared to larger particles when contacting the
cell [19].
2.3. Crystal Structure
The most common cause of the toxic effects of NPs while interacting with cells is the release of
metal ions [8]. The dissolution is influenced by crystallinity, crystal phase, surface strain, size, defects,
media composition and so forth. Since NPs have a greater fraction of atoms at the corners and edges,
this makes it easier for ions from the surface to break away from NPs due to high free energy [20].
For example, due to the variation in surface Gibbs free energy of different faces, the dissolution
kinetics of polar (0001) terminated ZnO NPs in pure water was higher than of non-polar (1010) crystal
surfaces [21]. In this context, He et al. [22] found that (0001) face had 3 times higher oxygen vacancy
abundance in the bulk than in (1010) surfaces.
Nano TiO2 containing amorphous phase was more soluble than crystalline TiO2 while pure
anatase NPs were more soluble than mixed anatase-rutile NPs [23]. Similarly, in an artificial media
at neutral (pH 7) and low (pH 1.5) pH, nano-anatase displayed greater solubility compared to
nano-rutile [24]. However, Gurr et al. [25] discovered that rutile type TiO2 NPs damaged DNA and
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triggered chromosome segregation and membrane changes, while anatase TiO2 NPs with the same
sizes were found to be non-toxic. It follows that the complexity of toxicity assessment should account
for other factors such as aggregation, shape, ROS formation and the complexity of bioenvironment and
bio interactions, where organic or ionic molecules may form soluble or non-soluble complexes with the
released ions that can control bioavailability.
2.4. Dispersibility and Aggregation
Because of the van-der-Waals forces, higher surface energy, and/or magnetic attraction, NPs show
a sharp tendency toward agglomeration and when used at a concentration of 1000 ppm, MONPs tend to
form aggregates [26]. The degree of aggregation influences biodistribution, biological and biomedical
activities of nanooxides. For example, Lousinian et al. [27] discovered that the small aggregate size
of ZnO NPs triggered high fibrinogen adsorption versus larger aggregate size due to the inverse
relation between aggregate size and surface area. It was also concluded that the aggregations of very
small MgОNPs (~5 nm) could reduce the efficiency of interaction with bacteria [28]. For that reason,
the engineered NPs are usually stabilized in colloidal suspension by organic or inorganic compounds
such as carbonate, cysteine or surfactants and they determine the surface charge of the whole system at
different pH values. When MONPs have not stabilized appropriately, the particles may aggregate due
to the colloid instability that could impede or slow their clearance and trigger eventual toxicity [29].
2.5. Surface Properties
The toxicity of the positively charged NPs is thought to be higher than that of negatively
charged because of the electrostatic attraction with the negatively charged cell membrane. However,
the positively charged NPs displayed an enhanced capacity for opsonization (adsorption of plasma
proteins) [30] and their interaction with antibodies, serum proteins and so forth, might change the
conformation of the adsorbed molecules leading to a change in their activity. There have been reported
a correlation between the surface charge and the cellular uptake because positively charge NPs had
higher mineralization rates in human breast cancer compared to negatively charged but both NPs
were internalized equally into human umbilical vein endothelial cells [31]. Xiao et al. [32] showed that
higher charged micellar NPs were massively incorporated by macrophage while NPs with a slight
negative charge indicate high tumor uptake and low macrophage clearance. Additionally, the surface
properties of MONPs are characterized by their zero-point of charge and acidity constant [18–33]
values that were closely related to the particle aggregation.
2.6. Photocatalytic Activity
The most agreed mechanism of photocatalysis is related to the formation of holes trapped into
surface defects and electrons localized into small hydrogen-rich areas. The charge separation minimizes
the chance for hole-electron recombination and increases the photocatalytic, electrochemical activity
and antibacterial effect [34]. The positive holes become trapped by water molecules from the air or
solution that is oxidized producing •OH radicals which are powerful reactive oxygen species (ROS).
The electrons in the conduction band can also trap oxygen which is reduced to form superoxide
(O2−• ) radicals that combine with H+ and produce peroxide radicals (•OOH) or hydrogen peroxide
(H2 O2 ) [35]. Some MONPs such as ZnO can generate superoxide, hydroxide radicals and singlet
oxygen while others can produce one, two or no ROS molecules [36] or even inhibit ROS production
induced by H2 O2 which suggests anti-oxidant potent [37].
2.7. Chemical Composition
Nanoparticle-induced toxicity is found to depend on the MONPs type which could be attributed
to NPs ability to generate ROS molecules and to release metal ions as mentioned above. For example,
ZnO, SiO2 , TiO2 and Al2 O3 NPs of the same size (around 20 nm) demonstrated different toxicity on
human fetal lung fibroblast (HFL1) [38]. After 48 h of treatment at concentrations of 0.25, 0.50, 0.75,
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1.00 and 1.50 mg mL−1 , MTT analysis showed that ZnO NPs were more toxic to HFL1 followed by
TiO2 , SiO2 and Al2 O3 in descending order. Other study demonstrated that copper and zinc oxide
NPs appeared to be more toxic to two human pulmonary cell types while titania, alumina, ceria,
and zirconia showed low to moderate toxicity without indicating a correlation between toxicity and
either specific surface area or equivalent spherical diameter [39];
2.8. Target Cell Type
Different cell target specificity commonly displays different metabolic activity and therefore,
different cell death mechanisms and sensitivity to MONPs exposure. When comparing the toxic effects
of nano SiO2 on human monocytes (THP-1) and human lung epithelial cells (L-132), it was found that
SiO2 NPs were more cytotoxic on THP-1 cell than on L-132 [40]. The cellular target specificities could
be attributed to the function of phagocytosis which characterizes monocytes but not lung epithelial
cells. Comparing the dose-dependent and time-dependent cytotoxic effects of different MONPs on
two cell types – alveolar (A549) and distinguished monocytes to macrophages (THP-1) cells, A549 cells
showed less sensitivity than THP-1 cell [39]. The higher sensitivity for macrophage responses and
their superior capability to take part in particle aggregates via phagocytic mechanisms were expected
to increase the macrophage responses to NPs.
3. Applications of MONPs in Biomedicine
To be used for a particular application, MONPs should meet certain requirements. For example,
the MONPs implemented as drug carriers should have kinetics complying with the requirements for
treating a certain infection and they have to be biodegradable to exclude further surgical intervention.
Although a broad spectrum of MONPs is available, only TiO2 , ZnO, CuO, ferric oxide (Fe2 O3 ) and
ferrous oxide (Fe3 O4 ) appeared as comparatively safe for mammals [41].
3.1. Internal Tissue Therapy
The implementation of therapeutics is usually closely related to the capacity to affect diverse
molecular signaling pathway that regulates the expression of growth factors, division, cell differentiation,
migration and apoptosis [36]. MONPs could penetrate the body by respiration, ingestion, through
the skin or by infusion or direct injection or transportation with nanofibers to a certain organ.
The great advantage of nanomedicine for disease therapy constitutes the potential to create a
nanocarrier structure with enhanced delivery efficiency due to the ability to translocate through
the cell membrane [4]. The benefits of developing nanocarriers as drug-delivery systems include
enhanced pharmacological activity, sustained and target delivery of more than one therapeutic agent,
stability and bioavailability [42]. Engineering materials allow for improving the specificity of the
nanosystems thus decreasing the side effects for patients. After entering the target cell, the toxicity
of a certain nanoparticle does not give a regular pattern of changes. A summary of the circulation
interactions and common mechanisms of MONPs induced cytotoxicity is shown in Figure 2.
The fate and cytotoxic effects of the MONPs may include:
•
•
•
•
•
ingestion by the phagocytic cells (monocytes, macrophages and dendritic cells) that could pose a
hazard to the cells [43,44];
opsonization [45] or enzymic degradation;
changes in cell membrane structure, integrity and disturbed function of its components influencing
the cell transport [25,46];
chromosomal segregations, aberrations, chromatin condensation and changes in the cell
replication rate [38,47];
hindered autophagy and macromolecules metabolization because of ruptured lysosomes which
could activate apoptotic caspase pathways [48];
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•
•
•
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disturbed production of energy through respiration and cellular metabolism by mitochondria
damage [49];
lower growth rate and cell division, structural changes and shorten lifetimes of microtubules of
the cytoskeleton and hampered intracellular transport [50–52];
generation of ROS and oxidative stress induces changes [47,53];
production of ROS likely leads to protein denaturation, DNA oxidation and membrane lipid
peroxidation which damages the cell integrity and influences the respiratory activity causing
eventually cell death. Besides, in physiological conditions, ROS produced mainly by mitochondria
mediate the intracellular signal transduction, regulate the protein phosphorylation and control
intracellular Ca2+ homeostasis [54].
Figure 2. Mechanisms of metal oxide nanoparticles (MONPs) delivery pathway and cell damage in
eukaryotic cells: (1) interaction with ions in circulation; (2) ingestion by phagocytic cells; (3) opsonization
or enzymic degradation; (4) internalization via endocytosis after extravasation to the extracellular
space or (5) membrane perforating and damage of its components and their function; (6) chromosomal
aberrations and changes in cell replication rate; (7) lysosome rupture; (8) mitochondria damage;
(9) lower growth rate, structural changes and shorten lifetimes of microtubules of the cytoskeleton;
(10) generation of ROS, oxidative stress and subsequent processes.
All the above-listed NPs’ triggered effects which damage the eukaryotic alter the cell adhesion,
division,
viability, differentiation, proliferation, migration, angiogenesis, and/or apoptosis. For that
reason, the identification of the exact molecular mechanisms of NPs influence and control on the cell
metabolism
and functions is of utmost importance. The mechanisms of particles’ movement in the
body
right should also be specified in detail to open up the possibility of exact focusing of NPs towards
target cells which will make their usage safer showing minimum side effects.
By targeting specific sites, NPs can reduce the overall dose of the medicament and thus to
decrease the undesirable side effects. One of the challenges in targeting therapy with NPs is to
the undesired interaction with other molecules, toxic effects on normal tissue and to increase
reduce
selectivity towards cancer cells or other target cells. This type of targeting therapy includes delivery
andtargeting nanocarriers linked with natural polymers such as polysaccharides, polyesters, DNA,
RNA, polypeptides, enzymes, proteins and so forth or synthetic polymer materials conjugated with
inorganic
NPs – silica, MOs, HAP, and so forth (Figure 3). The coating shell is usually hydrophilic and
makes the functionalized NPs compatible with bio-environment [55]. The surface coat determines the
overall size of the colloid particle and is very important for the biokinetics and biodistribution of NPs
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in the body [56]. The shells could contain biological bioactive molecules such as organic acids, chitosan,
gelatin or liposome coatings or polymeric materials like PEG, poly(vinyl alcohol) and so forth and
surfactants (SDS, sodium oleate, etc.) that could protect, enhance or give additional effect and/or direct
the NPs in the organism. The whole engineered system with a biological and non-biological origin that
treats prevents or diagnosis
a certain disease is called theranostics (diagnostic with target therapy).
‐
In that context, NPs should have a high loading capacity. The drugs could be covalently bonded to
functionalize MONPs, for example, HAP [57] or to establish electrostatic interaction with the charged
NPs. The medicaments linked to NPs could be anticancer, immunosuppressive, anticonvulsants,
anti-inflammatory, antibiotics, antifungals, antiviral and alternative
drugs. The ability of MONPs to
‐
localize the drug to the target cell may greatly enhance the use of these medicaments by reducing the
necessary dose and sparing the normal tissue toxicity. The shell of the nanohybrid material could not
only stabilize the MONPs but also eliminate the toxicity of NPs due to the formation of ROS, change
their stay in the organism and make them tissue-specific [58]. The whole NPs should demonstrate
good penetration
and accumulation in the target cell and once the carriers are incorporated in the cell,
‐
they should be able to escape endosomes.
Figure 3. Scheme of possible modifications of MONPs for biomedical applications.
3.1.1. Iron Oxide Nanoparticles
The magnetic properties of iron oxide NPs brand them suitable for magnetic separation of
biological products and cells, diagnostics and guides for site-specific drug delivery [59]. Fe3 O4 NPs
‐
are magnetic biomaterials that could be directed and concentrated by the external magnetic field and
to be removed once the therapy is completed [60]. In the bloodstream, iron oxides NPs are usually
subject to opsonization followed by recognition and elimination in the blood circulation [44]. The high
vascularization and permeability of iron NPs trigger their uptake by the reticular endothelial system
and make them recognizable by macrophages [43]. However, it is generally accepted that Fe3 O4 NPs
can kill cancer cells without compromising the regular cells. Since cancerous tissue is more sensitive
to heat damage in vivo, magnetic NPs can target heat specifically to tumors by alternating magnetic
fields, frictional or hysteresis heating [61]. This treatment is called hyperthermia. Hilger at al. [62]
injected supermagnetic NPs into immunodeficient mice with implanted breast adenocarcinoma cells
and after applying a magnetic field with 400 kHz frequency, the temperature within the tumor region
rose to 73 ◦ C. Based on the general presumption for penetration of NPs through various membranes,
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Hurbankova et al. [63] studied the impact of Fe3 O4 NPs on the vascular system of the respiratory tract
together with the inflammatory and cytotoxic parameters of bronchoalveolar lavage. The iron oxide
NPs, compared to the control, induce an inflammatory response, cytotoxic damage and respiratory
toxicity. The results further showed that Fe3 O4 NPs after 28 days of installation were eliminated from
the respiratory tract by the defense body mechanisms.
Iron is an important ion in all cells’ homeostasis. Due to the colloid instability of bare iron
oxide NPs, different modifications improve the stability and prevent the opsonization (adsorption
of plasma proteins) of NPs in blood circulation. The natural polymers such as the polysaccharide
chitosan showing hypoallergenic, antibacterial and hemostatic properties, were used for the synthesis
of chitosan-coated iron oxide NPs to develop drug delivery system to treat low bone mineral density
like osteoporosis or slackening of the prosthesis [64]. The authors revealed that the majority of
modified NPs were extracellularly located while the uncoated iron oxide NPs was predominantly
found intercellular. The chitosan linked NPs amplified osteoblast proliferation, distinction and viability
but at a concentration above 300 µg mL−1 , the increased internalization of iron oxide NPs by osteoblasts
induce apoptosis. Nevertheless, no concentration-dependent internalization of chitosan-coated NPs
was witnessed suggesting fewer dosage restrictions in clinical practice. Other hemolytic and cell
viability studies revealed that when iron oxide NPs were conjugated with BSA, improved specific
absorption rate values were observed due to the enhanced colloidal stability and prevention of NPs
aggregation [65]. Most hyaluronic acid (HA) drug conjugates with iron oxide NPs as a targeting moiety
had been developed for cancer chemotherapy as macromolecular products [66].
Magnetic nanotubes allow not only magnetic properties but also differential functionalization
of outer and inner surfaces which can be useful for magnetically assisted drug delivery. Yu et al [67]
functionalized porous iron oxide nanorods with folic acid for target delivery of low water-soluble
anticancer drug – doxorubicin (DOX) and found out that the presence of folic acid on the surface of
nanostructures increase the cytotoxicity of doxorubicin and the cellular uptake by HeLa cells. This effect
the authors attributed to the specific binding between folate receptors and folic acid which made these
nanocarriers suitable for targeted drug delivery.
Similarly, various attempts had been made to control aggregation, anisotropy, specific absorption
rate and so forth by doping MONPs not only with biomacromolecules such as HA, BSA, small interfering
RNA (siRNA), proteins or chitosan but also with different synthetic polymeric coatings. Feng et al. [12]
studied the in vivo and in vitro biological behavior of commercially available iron oxide NPs (10 and
30 nm) coated with PEG with positive surface charge and PEI which had an almost neutral charge.
PEI coated NPs exhibited severe cytotoxicity contrary to macrophage and cancer cells while PEG-coated
indicated no obvious cytotoxicity even at higher concentrations. Moreover, PEG-coated NPs were
capable to induce autophagy which may have a defensive role against the cytotoxicity of iron oxide
NPs. When drug-loaded, iron oxide NPs help to control the release of the medicament which could
reduce the side effects due to their lower dosage and minimize or prevent the degradation of the drug
because of the ability other pathways than gastrointestinal to be used [68].
Dendrimers are globular nanostructures with a core from MONPs or Au, encapsulating special
molecules (drug, gene or cancer imaging) in their internal voids. A dendrimer can also perform
solubility or dissolution enhancement and controlled delivery [69]. A schematic view of a typical
dendrimer with its important parts, that are core, branches, generation numbers, functional groups
and so forth, and dendrimer–cargo interactions are shown in Figure 4. Dendritic NPs such as PAMAM
(poly(amidoamine)) are widely explored for drug delivery agents in cancer and antiviral therapy,
gene delivery, medical imaging applications and vaccine delivery systems [70]. The quantity of the
entrapped molecules depends on the size and shape of the dendrimer and the size of its internal
cavities (Figure 4). These cavities are usually hydrophobic thus allowing for interaction with poorly
soluble drugs or charged molecules like siRNA [71].
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‐
Figure 4. General representation of a model structure of a dendrimer and possible dendrimer-molecular
cargo interactions.
‐
Polyvalent dendrimer-bearing
magnetic NPs as carriers for siRNA delivery evaluated in a
transgenic murine model of glioblastoma in vivo and in vitro promoted cytosolic release of endocytosed
cargo more resourcefully than their components, resulting in an effectual delivery of siRNA to cell
‐
cytoplasm over a wide range of loading doses [72]. The non-covalent attachment
of siRNA afforded
dendriworms the ability to hold flexibility in siRNA loading without reformulation. The drug-release
‐
rate depends on the type of the chemical bond between the drug and carrier, the structure and steric
effects of the conjugated NPs. The in vivo efficiency of anionic G4.5 PAMAM dendrimers with magnetic
‐
‐
NPs as drug carriers for low water-soluble antipsychotic drug-risperidone was evaluated for the
parameters of heart rate and brain development of Zebrafish larvae [73]. The most significant changes
were observed when larvae were treated with free risperidone, not with the dendrimer NPs. This may
indicate a decline in the side effects on the drug once administrated as a complex or decrease the effect
on its onset. The toxicity of PAMAM Khodadust et al. attributed to the dense surface amine groups [74].
Consequently, suitable surface modification with drugs, vitamins, antibodies, PEG or imaging agents
producing neutral or anionic surfaces can reduce the dendrimer biotoxicity. Other authors relate the
mechanism of toxicity of the micellar NPs with their surface charge because the neutral surface charge
has the longest circulation time and have lower uptake in the liver and spleen than positively and
negatively charged NPs [75].
3.1.2. Zinc Oxide Nanoparticles
Compared to normal cells, ZnO NPs showed selective cytotoxicity toward cancer cells in vitro and
in vivo [76,77]. ZnO itself presents certain cytotoxicity in cancer cells due to higher ion release in acidic
media and increased ROS induction and hence is used in cancer therapy. Evaluating the anticancer
‐
activity of ZnO NPs, Moghaddam et‐ al. [49] observed apoptosis triggered by extrinsic and intrinsic
‐
apoptotic pathways in MCF-7 cancer cell line. The authors reported cell cycle arrest, a 5-fold increase
‐
in stress-responsive kinase inducing apoptosis and depolarization of the mitochondrial
membrane
potential indicating that ZnO NPs apoptosis was mitochondria-dependent. In human keratinocytes
(HeCat) and epithelial cells (HeLa), commercial ZnO NPs and custom-built ZnO nanowires induced
acute cytotoxic effects prompting actin filament bundling and structural changes in microtubules
renovating them into rigid microtubules of tubulin [50]. ZnO NPs severely affect cell proliferation and
survival because of acute cytoskeletal collapse triggering necroses followed by late ROS-dependent
apoptotic processes.
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As discussed previously, ZnO NPs are attractive candidates for cancer drug delivery because
of their biodegradable characteristics [77]. Doxorubicin-ZnO nanocomplex was found to act as an
efficient drug-delivery system against hepatocarcinoma cells enhancing the chemotherapy efficiency by
increasing the intercellular concentration of doxorubicin [78]. Additionally, the nanocomposite showed
excellent photodynamic therapeutic properties. Hollow ZnO spheres loaded with paclitaxel and
functionalized with folic acid successfully induced cytotoxic effect against breast cancer cells in vitro
and in vivo by reducing xenograft tumors in nude mice [79]. As in iron oxide NPs, to enhance the
drug solubility, bioavailability and efficiency, Dhivya et al. [80] synthesized copolymer encapsulating
ZnO NPs with hydrophobic PMMA-AA loaded with a large amount of hydrophobic drug curcumin.
The results notified that the percentage release of curcumin was higher at pH 5.4 as opposed to pH
6 and pH 7.2. This fact is very important as the tumor cells are stable at about pH 6. Moreover,
the Cur/PMMA-AA/ZnO NPs showed exceptional cytotoxicity contrary to ACS cancer cells compared
to other bionanocomposite materials under similar experimental settings.
Since zinc participates in insulin synthesis, storage and secretion [81], ZnO NPs have also the
potential to be effective anti-diabetic or alleviates diabetic complications agents when stabilized or
conjugated with certain substances. In 2018, Hussein et al. [82] synthesized conjugated ZnO NPs
(20–27 nm) with naturally biodegradable and biocompatible polymer-hydroxyethyl cellulose (HES) by
a wet chemical process. With the purpose to attenuate the diabetic complications, male albino rats
were treated with the conjugated ZnO NPs. The results indicated that CPR, pro-inflammatory IL-1α
and ADMA levels increase meaningfully concomitant with a reduction in NO (signaling molecule
for regulating angiogenesis, protein kinase G activity, protein phosphorylation and other processes
connected with cell proliferation, migration and vasodilatation) level in the diabetic rats while ZnO
NPs supplementation suggestively attenuated these factors which make ZnO particles very promising
for enhanced selectivity towards atherosclerosis.
3.1.3. Titanium Dioxide Nanoparticles
TiO2 is a prevalent material for biomedical applications used mainly in bone and tissue engineering
owing to its ability to induce cell adhesion, osseointegration [83], cell migration and wound healing [84].
In vivo experiments with non-modified TiO2 upon 365 nm light irradiation indicated tumor growth
suppression in glioma-bearing mice together with higher mice survivability [85]. Nitrogen-doped
anatase NPs revealed higher visible light absorbance than neat TiO2 and at a concentration of
0.5 mg mL−1 resulted in 93% cell death of melanoma cells under UV light [86]. The authors also
found that depending on the type of cancer, the sensitivity of cancer cells to modified NPs may vary
significantly. However, the penetration of UV light through tissues is low and harmful and, therefore,
photodynamic therapy related to tissue overhealing is significantly limited [87].
When titania nanotube arrays with 100 ± 10 nm diameter were decorated with TiO2 NPs,
a significant increase in the specific surface and, therefore, loading efficiency of ibuprofen (an antiinflammatory drug) was observed [88]. The release efficiency of gentamicin-loaded anodic TiO2 was
compared for both nanotubes with and without a 2.5 µm thick coat of chitosan and poly(lactic-co-glycolic
acid) (PLGA) [89]. In contrast to uncoated NPs that release the drug after 2 weeks, the modified
nanooxide demonstrated extended-release up to 22 and 26 days for chitosan and PLGA, respectively.
In 2018, Masoudi et al. [90] prepared TiO2 NPs with inherited fluorescence properties and high
doxorubicin hydrochloride (DOX) loading capacity. In vitro cytotoxicity test on human osteosarcoma
(SaOs-2) and breast cancer (MCF-7) cell lines revealed higher anticancer efficacy (lowered IC50
concentration by 5.5- and 3 fold for MCF-7 and SaOs-2, respectively) and better imaging for intercellular
tracking of DOX loaded NPs relative to free DOX. In another study, diamond-shaped TiO2 NPs were
functionalized with PEG chains and loaded with DOX [91]. In acidic conditions associated with cancer
cells, DOX was almost entirely released. During in vivo tests, Bulb/c mice bearing H22 tumors showed
smaller tumor volumes when treated with the complex drug nanocarrier as compared with DOX-free
treated groups.
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It follows that MONPs in combination with target molecules and drugs offer an efficient platform
for delivery of therapeutics thanks to either simple delivery or other active release mechanisms.
In that way, fewer adverse effects in health cells occur while the amount of the therapeutic agent is
significantly lower. By assessment of a mixture of different drugs and therapies encouraging results
have been reported.
3.2. Immuno-Therapy
NPs can interact with different components of the immune system and thus enhance or inhibit
its function [92]. NPs can be specifically designed to suppress (for example anti-inflammatory) and
stimulate (i.e. vaccine) immunity. The first step of recognition of NPs by the immune system as foreign
materials is the adsorption of blood proteins which type and quantity determine the fate of NPs by
interaction with other molecules. If NPs bind to the cell surface, they can initiate signaling processes
triggering immunogenicity or toxicity. After activation, the immune system can release cytokines that
act as a mediator of systemic and local inflammatory and hypersensitive reactions [11]. For instance,
since ROS can function as a second messenger and modulator in immunity, ROS from TiO2 NPs can
activate downstream pro-inflammatory effects and avoid the innate immunity in macrophages [37].
These observations suggest that MONPs with different surface reactivity can modulate the immune
function trough ROS-activated pathways. Attachment or encapsulation of charged molecules such as
enzymes, amino acids and so forth to NPs, can protect them from recognition by the immune system
and prevent the adsorption of opsonins. Besides, careful design of various components can reduce the
toxicity of these formulations [93].
3.2.1. Iron Oxides Nanoparticles
Iron oxide NPs are also used as potent carriers for vaccine delivery with improved therapeutic
effects. A way used to achieve successful inhibition of viral replication was the incorporation of small
interfering RNA (siRNA) onto nanocarriers [94]. Other strategies include the use of transactivator
or transcription protein as an adjuvant to target cell for intercellular delivery of MONPs or MONPs
inhibition of viral glycoproteins that are vital to attach the host cell and thus inhibit the infection [95].
Iron oxide NPs were used as undercover carriers to deliver anti-retroviral drugs to a latent form
of HIV while reporting the localization of drugs owing to their contrasting properties [96]. A complex
anti-HIV drug, enfuvirtide that is unable to cross the blood-brain barrier (BBB), was able to cross it
when the drug was loaded on a PMA amphiphilic polymer-coated over iron oxide NPs [97]. NPs have
been shown to stimulate an immune response, including activation of antigen-presenting cells and
induction of cytokine and chemokine release [98]. When captured by macrophages, supermagnetic iron
oxide NPs could promote the pro-inflammatory phenotype in macrophages [99]. Shevtsov et al. [100]
invented nanovaccine of superparamagnetic iron oxide NPs coated with recombinant heat shock
protein 70 (Hsp 70) antigenic peptide that was able to stimulate tumor-specific T cell response in glioma
bearing rats. By facilitating antigen trafficking to antigen-presenting cells (APC), a delayed tumor
progression and increased overall survival were observed. In M2-polarised macrophages that are
principally involved in wound healing, resolving inflammation, angiogenesis and tissue remodeling,
internalized iron oxide NPs coated with dimercaptosuccinic acid, aminopropyl silane or aminodextran
(the later as a prospective contrast agent and biomolecule delivery) showed no cell toxicity [52].
MONPs are also used to enhance the protective and long-lasting immune response of the safer
but less immunogenic subunit vaccine than live attenuated vaccines. Citrate-coated MnFe2 O4 NPs
with protein corona of the fusion protein (CMX) composed of Mycobacterium tuberculosis antigens were
proved to be capable of aiding the generation of the specific cellular immune response (T-helper 1,
T-helper 17 and TCD8) [101]. Besides bacteria antigens, Fe2 O3 NPs have been utilized in the creation of
virus-like particles (VLP) that proved their exceptional use as vaccines, gene-carrying nanocontainers,
MRI contrast agents and drug delivery vectors [102]. In this approach, bacterial viruses had been used
not only as phages conjugated with functionalized (with carboxyl and amino groups, respectively,
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for better loading) MONPs for antimicrobial purposes [103] but also as a scaffold to carry more Fe2 O3
NPs that can powerfully bind with cancer cell surfaces than Fe2 O3 NPs themselves [104]. Moreover,
antibody and antibody-like conjugates to NPs can be bi-specific complexes with multiple modes of
action including the delivery of toxins or agents that kill tumor cells; inhibition of two ligands or
receptors; and crossing of two receptors [105]. However, there are many gaps in understanding the
immune response that MONPs induce.
3.2.2. Zinc Oxide Nanoparticles
ZnO NPs have been reported to trigger cytokine and chemokine production which are used
as biomarkers for immunotoxicity. They were found to exhibit adjuvant outcomes through diverse
mechanisms including activation of the innate immune response, augmentation of the antigen uptake
by antigen-presenting cells and regulation of cytokine network [44]. For instance, exposure of dendritic
cells to ZnO NPs with negative zeta potential was found to upregulate the expression of CD80 and
CD86 and stimulate the release of IL-6 and TNF-α without any cytotoxic effects [106]. Additionally,
ZnO NPs were found to induce the degradation of IκBα that is NFκB inhibitor and thus to stimulate
NFκB signaling [107]. Specifically designed ZnO tetrapod NPs demonstrated the effective suppressive
function of HSV-2 genital infection in female Balb/c mice when used intravaginally [108]. The prior
incubation of HSV-2 with ZnO NPs promoted local immune response similar to the infection but
with less clinical manifestations and inflammation. The suppressed infectivity the authors attribute to
facilitate intracellular delivery of viral antigen into APCs and, therefore, activated antigen-specific
adaptive immunity.
Simultaneously, chemically synthesized NPs were found to be more toxic to cells and living tissue,
whereas green synthesized NPs showed lesser toxicity probably because of phytochemical capping
that may suppress the production of ROS [109] and thus the inflammatory reactions. Thatoi et al. [110]
evaluated the anti-inflammation activity of bio-reduction synthesized ZnO NPs from mangrove
plants namely Heritiera fomes and Sonneratia apetala by the capability of different NPs to impede
protein denaturation trough blocking heat-induced albumin denaturation. The maximum inhibition
activity (63.26 µg mL−1 ; IC50 ) was observed for S. apetala synthesized ZnO NPs followed by H. fomes
synthesized ZnO NPs (72.35 µg mL−1 IC50 ) which values were close to that of conventional drug
diclofenac (61.37 µg mL−1 IC50 ). In a dendritic cell line, PLLA microfibers coated with ZnO nanowires
were observed to significantly induce inflammatory cytokines like IL-10, IL-6, IL-1β and TNF-α while
introducing a tumor antigen [111]. The nanocarrier including ZnO enhanced the infiltration of T cells
into the tumor tissue compared to mice immunized with PLLA fibers directly conjugated with tumor
antigen. Recently, Hu et al. [112] showed an enhanced antitumor therapeutic effect in in vivo animal
models injected with 4T1 tumor cells, especially in a combinatorial administration with ZnO NPs
and doxorubicin (DOX). It was demonstrated that ZnO NPs promoted Atg5–regulated autophagy
flux suggesting that autophagy induction by ZnO reinforces cancer cell death by increased Zn ion
release and ROS generation when combined with DOX. It can be summarized that the use of MONPs
increases the effectiveness of immunotherapy because of the specific characteristics of these nanocarrier
systems. The targeting ability of NPs can be changed by different modifications including therapeutic
agents, bioactive moieties or drugs. The research on the activities and effects of these NP systems is
widely studied and still ongoing. Combined immunotherapy with other treatment methods such as
chemotherapy, photothermal therapy and so forth, also gained attention. However, challenges remain
due to lack of understanding about in vivo behavior of NPs that can act both as immune-stimulating
adjuvants and/or delivery systems to activate antigen processing.
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3.3. Diagnosis
MONPs have been extensively used for diagnostic purposes due to their fluorescent or magnetic
behavior. The advantages of nano-oxides allow a more precise visualization and reliable quantification
of disease viability and progression.
3.3.1. Quantum Dots for Labeling
Various biomedical targets such as cancer cells, stem cells, bacteria or individual molecules could
be labeled with highly fluorescent NPs. Quantum dots (QD) are highly fluorescent and photostable
colloidal semiconductor NPs with 2–10 nm diameter [113] that is a useful tool in in vivo imaging
cellular structures and events. In such a way monitoring of cell migration, retention in target sites or
evaluation of viability can be done. When excited, QDs emit light with sharp and symmetrical emission
spectra and high quantum yield. Their main characteristics are chemical and photostability, catalytic
properties and controllable electron/ion transfer effect. However, the safe use of these QDs when
introduced in the organism is of prime importance. For that reason, Wierzbinski et al. [114] investigated
iron oxide NPs used as labeling agents for in vitro investigations of human skeleton myoblast cell line
and in experimental animals after intramuscular and intracardial administration. The study showed
no impact of labeling myoblasts with dimercaptosuccinate (DMSA) coated iron oxide NPs on their
differentiation comparing to non-labeled cells. The gene expression levels of FTL (a biomarker for
aging) encoding the light chain of the transferrin receptor that transfers the extracellular iron ions
into the cytosol, was similar for both treated and untreated cells. Although influencing some gene
expression, DMSA-coating NPs did not disturb stem cell homeostasis. Other authors improved cell
labeling functionalization of ZnO NPs by applying silica coating which was conjugated with biotin
(vitamin) amino group [115]. When avidin-attached nerve cells were exposed to biotin-conjugated
ZnO NPs quantum dots with a size of 125 nm, green emission from the cells was observed and no
emission was observed from silica-coated ZnO NPs without biotin. Additionally, biomarkers of NPs
like fluorescent dyes were used for in vivo ocular imaging [116]. Further research should be done
to elucidate the individual QDs transport, interactions, toxicology and fate in certain tissues, organs
and organisms.
3.3.2. Contrast Agents for Magnetic Resonance Imaging
Magnetic resonance imaging (MRI) is a widely used non-invasive clinical practice due to its good
soft-tissue contrast, high spatial resolution, 3D anatomical information and non-ionizing radiation.
Magnetic imaging techniques permit different molecular changes related to the inception and progress
of pathological states to be counted and to provide timely diagnosis and prognosis of diseases such
as cancer [117]. The contrast agents used in magnetic resonance imaging (MRI) are generally based
on either iron oxide NPs or ferrites. Polymers are the most extensively used stabilizing materials for
iron oxide NPs. Together they form negatively imaging agents producing a signal-decreasing effect
and the persistent effects from circulating NPs delay MRI by 24–72 h. When cancer cells of solid
tumors are examined, in contrast to other tissues, the poor lymphatic drainage aids the entrapment
of NPs. To increase signal sensitivity and enhance MRI diagnostics [118], alloy-based nanomaterials
forming compounds known as ferrites (for example Zn-ferrite) that increase the net magnetization of
NPs [119], were also used. Additionally, magnetoliposomes consisting of single iron oxide NP, vesicles
of water-dispersible iron oxides or PEG encapsulated iron oxides core and covering single phospholipid
bilayer [120] could increase the imaging contrast due to particle monodispersity. The ability to combine
iron oxide NPs with other metals or to form core-shell iron oxide NP structures helps in improving the
diagnostic information.
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3.4. Nano-Oxides in Dentistry
Eliminating the bacterial infection in the root canal system is the decisive goal of endodontic treatment,
preventing microorganisms from impairing periapical healing. For that reason, Javidi et al. [121] examined
ZnO NPs for antibacterial root canal sealer by microleakage measurements at 3, 45 and 90 days intervals
to check their stability and sealing properties. The microleakage increased with an increase in the
calcination temperature (from 500 to 700 ◦ C) because of an enlargement in the size of NPs and therefore,
decreased effective surface. All prepared groups of ZnO NPs samples exhibited less microleakage in
comparison with commonly used ZOE sealer. Another research group [122] synthesized more porous and
spongier ZnO/MgO NPs for polycarboxylate dental cement used in clinical dentistry for luting agents,
orthodontic attachments, cavity lining and bases and restoration for teeth. In contrast to conventional
zinc polycarboxylate cement, ZnO and MgO nanostructured and ZnO/MgO nanocomposite cement
had increased mechanical strength and comparable setting time for preparation of the dentin cement
to those obtained by commercial samples. Antibacterial nanofilms deposited on nanostructured
electrochemically deposited TiO2 NPs on the surface of NiTi arches were evaluated in vivo for their
histopathological, genotoxic and cytotoxic effects in Long-Evans rat [48]. After deposition, the coating
was susceptible to degradation in PBS solution when ingests were absorbed and TiO2 NPs entered
the bloodstream producing a toxic effect on the organs with the first step being the liver parenchyma
which resulted in extensive lesions. When internalized, TiO2 NPs accumulated in the lysosomes which
led to rupture and release of their content that subsequently activate apoptotic caspase pathways.
To generate novel biomaterial aimed at denture base that could obstruct the adhesion of
microorganisms to their surface and therefore, to decrease the growth of denture stomatitis,
Cierech et al. [123] modified polymethyl methacrylate (PMMA) with ZnO NPs. The increased
hydrophilicity and hardness with absorbability explained the reduced microorganisms’ growth
on the denture base. The antifungal properties increased with increasing the content of ZnO NPs
in the polymer. Similarly, a thin layer of nanostructured TiO2 film of the surface of Co-Cr alloy
revealed significant antifungal activity under UV-irradiation which can be considered in the future
against denture stomatitis [124]. Nanotechnology offers enhanced effectiveness of already available
technologies since it will be cost and time saving whilepreventing the patient from adverse side effects.
3.5. Nano-Oxides in Hard Tissue Regeneration
The bioactivity of the conventional titanium-based materials applied in orthopedic and dental
implants is insufficient in terms of bone-implant osseointegration. It is widely known that the
osseointegration of implants was enhanced if the surface topography and morphology of the natural
tissue mimicked by implant surface. Most of the extracellular protein, structures and cell sizes vary
between 10 and 100 nm. However, the implant material is not only recognized by targeting adherent
cells but also from the host immune system that triggers immune reactions which may eventually
determine the implant life. To increase cytocompatibility and decrease the macrophage activity,
Li et al. [125] synthesized magnetron deposited cerium oxide NPs on titanium surface and examined
the biological response and the underlying mechanisms of new bone formation in vitro and in vivo.
The prepared oxide NPs were in mixed Ce3+ /Ce4+ valence state and the increase in the surface Ce3+ /Ce4+
ratio promoted new bone formation and mineralization. The lower surface Ce3+ /Ce4+ ratio had high
superoxide dismutase (SOD) and low catalase mimetic activities that regulated the production of ROS.
Additionally, the surface Ce3+ /Ce4+ ratio was found to modulate the balance of anti-inflammatory and
pro-inflammatory cytokines in macrophages. It was concluded that the mixed-valence cerium oxide
NPs had the potential to induce bone regeneration without the need of any exogenous osteogenic
inducer. Even in the absence of osteogenic supplements, nanocomposite scaffolds of glass foams
containing nanoceria demonstrated enhanced collagen production and osteoblastic differentiation of
human mesenchymal stem cells (HMSCs) compared to scaffolds without nanoceria [126]. The authors
assigned the enhanced production of collagen of HMSCs to the incorporation of nanoceria that acted as
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an oxygen buffer regulating the differentiation of HMSCs. However, the effects of the released cerium
oxide NPs in the body are still unknown.
TiO2 is extensively used for dental and orthopedic applications because of its biocompatibility
and good mechanical properties. The morphofunctional reactions of the immortalized line of human T
lymphocyte cells (Jurkat leukemia cells) to short-term in vitro exposure to rough micro-arc oxidated
nano TiO2 coatings were examined by Kan Hlusov et al., in 2018 [127]. The metal-ceramic TiO2 film did
not prompt apoptosis or necrosis in T-cells.‐ However, the cells exposed to TiO2 coating with surface
roughness value Ra > 2.2 µm exhibited
μ a progressive decrease in viability and total suppression of IL-4
dependent mechanism
of T-cell survival. The roughness
of TiO2 dielectric surface-induced electrostatic
‐
‐
‐
potential, which was capable of varying the molecular genetic features and viability of leukemia T-cells
via mechanisms discrete
‐ to ROS generation. Our research group found out that electron beam surface
treatment of titanium alloy not only increased the surface micro- and nano roughness [128]‐ but also
decreased the surface hardness of the magnetron sputtered TiN with overlaying TiO2 coating bringing
it closer to that of trabecular bone and human teeth and decreasing the elastic modulus mismatch
between the bone and implant [129]. Moreover, nanostructured arc PVD deposited TiN with overlaying
‐
glow-plasma discharge deposited TiO2 coating
indicated an increase in MG63 cell viability by almost
100% after 24 h relative to the bare Ti6Al4V alloys [130]. The coated samples displayed better cell
attachment and cytocompatibility with no negative effects on MG63 cells (Figure 5).
Figure 5. A strategy to improve osteointegration by applying inorganic nanostructured TiN/TiO2
coatings with enhanced surface characteristics as opposed to bare Ti6Al4V alloy.
Similarly, TiO2 nanotubes encourage interaction with osteoblasts by enhancing the contact area of
the bone cell-implant.
TiO2 nanotubes with diameters 30, 70 and 100 nm respectively, were investigated
‐
in vivo and compared with machined titanium implants for their bone-implant contact
and osteogenic
‐
gene expression levels [131]. The optimum size of the nanotubes for rapid osseointegration was found
to be 70 nm in diameter probably because of its similarity with the nanostructure of natural bone.
It follows that by altering nanoscale dimensions, nanotubes were able to control cell fate and interfacial
osteogenesis. Moreover, after comparing the bone mineral density and the volume of newly shaped
bone, those around the propolis modified TiO2 nanotubes with a diameter of 60–90 nm were much
higher than formed around individual TiO2 nanotubes at 1, 2, 3 and 4 weeks after implantation [53].
Collagen that is synthesized by differentiated osteoblast was found to be well-formed around the
‐
‐
propolis-modified
nanotubes. The expression
of inflammatory cytokines (TNF-α) displayed the
‐α
complete tissue around discrete TiO2 nanotubes but partially expressed around propolis modified
TiO2 nanotubes. It is summarized that the identification of the influence of the specific surface features
of biomaterials influencing the cell adhesion, proliferation or differentiation is difficult to be defined
because of the complexity of the surface factors and cell mechanisms influencing their interaction.
‐
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3.6. Nano-Oxides for Wound Healing
MONPs are used not only in hard tissue reconstruction but also in polymeric nanofibers to enhance
the overall properties of the composite scaffold for skin tissue engineering as wound dressing materials.
Wound healing follows a complex sequence of events including enhanced accumulation of growth
factors, proliferation of fibroblasts, and extracellular matrix synthesis that leads to re-epithelization.
It has been proved that ZnO NPs cannot pass through the skin [132] and slowly solve in an aqueous
solution as ions [133]. However, ROS generated by MONPs play signaling and regulatory roles in tissue
engineering. It was found out that ROS assessed not only an antiseptic role in wound healing but they
also participated in wound-to-leucocyte signaling in tissue [134]. For that reason, Augustine et al. [135]
investigated the effect of ZnO NPs concentration incorporated in electrospun polycaprolactone (PCL)
non-woven membrane on the cell proliferation of adult goat fibroblast cells. The results pointed out
that the PLC membranes containing 0.5 and 1% ZnO NPs enhanced cell proliferation more than neat
PLC-membrane. Another study also reported good fibroblasts proliferation results when ZnO NPs with
concentration up to 1% was incorporated in PMMA fibers and films [136]. Recent studies demonstrated
that the in vivo used microporous Ag/ZnO NPs loaded with chitosan accelerate significantly the
initial state of the wound healing process in mice [137]. The composite dressings showed a long time
moisture retention, enhanced blood clotting capability, high antibacterial activity against pathogenic
bacteria and denser collagen deposition properties compared with either pure chitosan dressing or ZnO
ointment gauze. Because not only ZnO NPs but also TiO2 NPs are proven to participate in increased
ROS production in bioenvironment, they were recently used in novel cutaneous would treatments.
TiO2 -impregnated biodegradable zein-polydopamine-based nano-fibrous scaffolds demonstrated
better-quality adhesion, proliferation and relocation of cells during in vitro tests [138]. Bacterial
cellulose (BC–temporary skin substitute) loaded TiO2 nanocomposites (with inherent antimicrobial
activity) were proved to participate in healing progression forming healthy granulation tissue and
re-epithelization in deep partial-thickness burns in case of mice model [139]. BC-TiO2 nanocomposite
treatment promoted suitable healing through fibroblast migration and suitable growth of epithelial
cells along with blood supply restoration forming new blood vessels.
3.7. Nano-Oxides Used as Biosensors
Nanobiosensors base on a ligand-receptor binding that evokes a reaction in the signal transducer.
According to their detection principle (signal measuring), some authors [140] classify the nanobiosensors
to piezoelectric, electrochemical, semiconductor, optical and calorimetric and all of them transferred
the information into electrical signals. The electrode material is crucial in the fabrication of highperformance electrochemical sensing platforms detecting target molecules using different advanced
analytical principles [141]. The nanobiosensors can be enzyme-based, genosensors, immunosensors,
cytosensors, and biosensors for the detection of small molecules. The main mechanisms of biosensing
are presented in Figure 6.
Small molecule electroanalysis is used to determine the presence of H2 O2 , glucose or dopamine.
For example, α-Fe2 O3 NPs in the form of cubes synthesized of hydrophobic iron-containing liquid
under hydrothermal conditions was used as a glucose biosensor material for non-enzyme catalytic
oxidation with high sensitivity and fast response [142]. MO nanostructures have been widely used in
the immobilization of enzymes because of strong adsorption capability, enhanced electron-transfer
kinetics and improved biosensing characteristics [143]. The enzyme-based biosensors contain
a thin layer of the immobilized enzyme on the surface of the working electrode. For instance,
the immobilization of the enzyme lactase detecting dopamine depends on the morphology of NPs
used [144]. Comparing three kinds of phytic acid modified silica NPs with different morphologies;
spherical, rod-like, and helical, for efficiency in dopamine detection, the best electrochemical
performance was defined for the helical-shaped NPs. TiO2 NPs on silica sol-gel modified gold
electrode immobilized with the enzyme lactate dehydrogenase were used as a biosensor for
determining lactic acid with a detection limit of 0.4 µmol L−1 and small Michaelis–Menten constant
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(Km app = 2.2 µmol L−1 ) [145]. Because of the large surface area and biocompatibility, ZnO nanorods
functionalized with glucose oxidase enzyme have been exploited as a miniaturized biosensor for
intracellular glucose measurements [146]. These nanostructured electrodes offer a biocompatible and
electroactive surface for enzyme immobilization with enhanced orientation and biological activity [147].
If the nanostructure is porous the active surface for protein binding increases and it provides a protective
environment for the enzyme to retain its activity and stability [148].
Figure 6. The illustration represents different mechanisms of biosensor activity; these bioreactions are
carried out in a controlled environment on the immobilization platform. Depending on the generated
signal, different transducing platforms are utilized to convert it into an electrical signal.
In the genosensors, single strained DNA fragments are immobilized on the electrode surface
while the immunosensors are based on the specific antigen-antibody recognition. The conventional
DNA detection
α‐ techniques mostly use fast electrochemical biosensors because of their‐high specificity,
portability and low cost [149]. The use of nanostructured materials allows for reducing the
‐ size, reagent
and sample consumption and development of a microfluidic genosensing system with increased
sensitivity. For example, CeO2 nanoshuttles-carbon nanotubes showed a remarkable synergistic effect
‐
enhancing the immobilization of DNA and, therefore, enhancing the sensitivity of target DNA detection
‐
via hybridization [150]. Another work combined the strong adsorption ability of Fe2 O3 microspheres
to DNA and the excellent conductivity of self-doped polyaniline nanofibers (SPAN) on carbon ionic
liquid electrode for electrochemical impedance sensing of immobilization and hybridization of DNA.
The proposed approach had a wide detection range, low detection limit (2.1 × 10−14 mol L−1 ) and
‐ discriminated against the target DNA from mismatch sequences [151].
successfully
The immunosensors employ either ‐antibodies or antigens as bio-recognition ‐elements usually
in combination with electrochemical transducers [143]. The sensing total performance depends on
the density of the immobilized antibodies/antigens which
makes
MONPs appropriate for improving
‐
μ
the functionality
− of the sensor. ZrO2 NPs on reduced graphene oxide had been functionalized using
μ
3-aminopropyl triethoxy saline (APTES), electrophoretically deposited on ITO coated glass and further
biofunctionalized with proteinaceous biomarker CYFRA-21-1 secreted in higher concentration in
oral cancer patients [152]. This immunosensor platform exhibited a wider linear detection range
‐
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(2–22 ng mL−1 ), excellent sensitivity (0.756 µA mL ng−1 ) and low detection limit (0.122 ng mL−1 ).
The shelf life of the immunoelectrode was found to be equal to 8 weeks. Upon immobilizing with
rabbit-immunoglobulin antibodies and bovine serum on nanostructured CeO2 film onto ITO coated
glass, fungal toxin-ochratoxin-A could be detected [153]. The immunoelectrode exhibit enhanced
characteristics in the linear range (0.5–6 ng dL−1 ), low detection limit (0.25 ng dL−1 ), rapid response
time (30 s), higher sensitivity (1.27 µA ng−1 dl−1 sm−2 ), and high value of the association constant
(Ka = 0.9 × 1011 L mol−1 ). Gold/ZnO nanocomposite films were employed to enhance the performance
of surface plasmon resonance (SPR) for tumor marker detection [154]. The linear range of response
to carbohydrate antigen extended from 1 to 40 U mL−1 and the limit detection fell to 0.025 U mL−1 .
The charged intensity of Au/ZnO sensor was increased by nearly 2 folds of Au/Cr layers whereas the
detection limit decrease 4 times than Au/Cr layers.
The cytosensors recognize antibodies, receptors, glycans or other molecules overexpressed on
the cell membrane of a target cell. For example, TiO2 nanowires functionalized with monoclonal
antibodies and immobilized on gold microelectrodes through mask welding were demonstrated as
sensitive, specific and rapid in the determination of bacteria Listeria monocytogenes for concentrations at
low levels of 102 cfu mL−1 for 1 h without interfering with other food-borne pathogens [155].
The photoelectrochemical assays always have advantages of both optical and electrochemical
detection [156]. The method could detect biomolecules such as glutathione at relatively very low
applied potentials using porphyrin-functionalized TiO2 NPs. Similar porphyrin-functionalized ZnO
NPs were advanced for the detection of cysteine with broad linearity in the range of 0.6–157 mmol L−1
in physiological media. The ability of nanobiosensors to be easily displaced and deformed in response
to very low forces of about 10 pN makes them sensitive enough to detect even the breaking of individual
hydrogen bonds [157]. It is clear that the key features of MONPs like high sensitivity and selectivity,
fast response and recovery times, reversibility, integration in different scales make these substances
suitable for the monitoring infection diseases, the pharmacokinetics of drugs, detecting of biomarkers
(cancer and disease), small molecules and so forth.
3.8. Antimicrobial Nano-Oxides
The interaction between NPs and bacteria usually triggers toxic effects that are exploited for
antimicrobial applications in industries such as food or agriculture. On one hand, the increasing
problem with AB resistance strains due to the transfer of AB resistance genes between bacteria
could be overcome by using bactericidal NPs that substitute certain conventional antibiotics. On the
other hand, the number of new fungal infections in immunocompromised patients and plants is
increasing throughout the whole world. Candida albicans is the most common yeast colonizing the skin,
reproductive system and gastrointestinal tract [158]. NPs interfere with different cellular processes of
the pathogens and the emergence of resistance is less likely to occur [159]. The antibacterial activity
of MONPs depends on mixture concentration and pH, size, distribution, and agglomeration of NPs
and displays dose- and time-dependent efficiency. Yamamoto et al. [160] demonstrated that when
the suspension of powder MO was concentrated enough, the larger the specific area, the better the
antibacterial activity. Also, the increase of speed of agitation of the dispersion of NPs in suspension
increases the death of bacteria indicating the importance of frequency of electrostatic contact and its
intimacy between NPs and cells [161]. The factors that influence the sensitivity of the bacteria to oxide
NPs are the synthesis parameters of NPs, structure of bacterial cell wall, and degree of contact with the
bacterial cell [162]. Figure 7 summarizes the main mechanisms of bacterial cell damage triggered by
the presence of MONPs.
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NPs as antimicrobial agents are highly promising because they have numerous modes of action
but the major lethal pathways usually occur simultaneously. It is generally accepted that most of
the MONPs exhibit bactericidal properties by generating ROS or releasing metal ions. For example,
the photocatalytic toxicity was found to induce lipid peroxidation under near UV-lamp causing
respiratory dysfunction and death of E. coli [163] and when combining with other nanomaterials like
Ag particles, synergetic antibacterial effects were observed [164]. NPs affect also the cell membrane
potential and integrity [165] and change the metal ion uptake into the cells followed by depletion in
adenosine triphosphate (ATP) production and DNA replication [166].
Figure 7. Mechanisms of bacteria cell damage by MONPs, illustrating the possible MONP interactions
with the bacterial cell wall, membrane components, DNA, enzymes and other proteins and the influence
of external ROS on membrane integrity, protein synthesis and functioning. Different MONPs may
cause toxicity via one or more of the described mechanisms.
The modern alternative for the production of MONPs from environment-friendly, non-toxic
and save reagents could use inactivated plant tissue, plant extracts, extrudates and other parts of
the living plant organism. The biosynthesis involves reducing metal ions or oxides with the help of
phytochemicals such as polysaccharides, reducing sugar, amino acids, alkaloids, vitamins, terpenoids,
saponins and other plant substances [167]. Except for different metal salts and reducing agents,
the biosynthesis of MONPs using plant extracts involves stabilizing or capping agents for better size
control of MONPs, prevention of aggregation [168], and, therefore, improvement of the biological
potential of NPs. Other organisms that are used for “green” methods of MONPs synthesis are bacteria,
fungi, yeasts, and algae but the plant extract mediated biosynthesis was found to be more stable
and faster than microbial synthesis [169]. The rate of synthesis of NPs was related to the reaction
and incubation temperature with the following tendency: the higher the temperature, the faster the
rate and the smaller the average size of the particles. The natural synthesis is environment-friendly,
cost-effective, taking less time and does not require costly equipment and precursors [170]. Recent
works focusing on the green synthesis of MONPs and their antibacterial effect are tabulated in Table 1.
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Table 1. Type, size, characteristics of the biosynthesis and activity of the MONPs against different tested microorganisms.
MO
Precursor
Biosynthesis
NP Size [nm]
Biological Activity/
Effect/Outcome
Tested Organism
Ref.
Biosynthesis of MONPs from plants
The highest inhibition zone was observed for K. pneumonia,
the lowest for S. aureus, and moderate for S. pneumonia;
The bactericidal activity was almost equivalent to that
of tetracycline.
[171]
-
Durable antibacterial activities against both S. aureus and E. coli
on nano colored wool fabrics.
[172]
-
At 5 mg concentration CuO NPs indicated effective
antimicrobial activity against gram-positive bacteria;
In this concentration, CuO NPs were much more effective
against Klebsiella and B. subtilis than standard drug ampicillin.
[162]
At a concentration of 512 and 1024 µg, E. coli strain was more
susceptible to Cu2 O NPs than Acinetobacter baumannii.
The zones of inhibition of cotrimoxazole and meropenem were
larger for both bacteria than that induced by Cu2 O NPs.
[173]
-
Significant antifungal activity against A. niger and M. piriformis
but more active against M. piriformis.
[174]
-
Moderate antimicrobial activity when compared to the
reference drug;
Higher bactericidal activity against E. coli and moderate
against S. aureus.
[175]
Definite antibacterial activity against all tested bacteria
without significant differences except in the case of S. enterica
where the bactericidal effect was lower in all 5, 10, 15 and
20 mg mL−1 concentrations.
[176]
Maximum zone of inhibition against gram-negative E. coli and
minimum zone against gram-positive Enterococcus sp.
[177]
CuO
CuS O4
Leaf extract of Eichhornia crassipes
20–22
S. pneumonia—S. aureus,
K. pneumonia
CuO
Cu(O2 CCH3 )2
Stems of Seidlitzia rosmarinus ashes
8–40
S. aureus, E. coli
16.8
G− (E. coli) and G+
(B. subtilis, S. aureus and
Klebsiella) bacteria
CuO
Cu(NO3 )2 ·3H2 O
Aqueous leaf extract of Abutilon indicum
-
-
Cu2 O
CuSO4 ·5H2 O
Aqueous leaf extract of Callistemon viminalis
423
E. coli,
Acinetobacter baumannii
α-Fe2 O3
γ-Fe2 O3
Fe(NO3 )3 ·9H2 O
Leaf extract of Platanus orientalis
38
Aspergillus niger,
Mucor piriformis
Fe3 O4
FeCl3
Seeds, leaves and fruits of Lagenaria siceraria
30–100
E.coli, S. aureus
Fe3 O4
FeSO4
Flower sheath extract of Musa ornate
43.69
S. aureus,
Streptococcus agalactiae,
E.coli, Salmonella enterica
ZnO
Zn(O2 CCH3 )2
Plant extract of Passiflora caerulea
30–50
Klebsiella sp., E.coli,
Enterococcus sp.,
Streptococcus sp.
-
-
-
-
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Table 1. Cont.
MO
Precursor
Biosynthesis
NP Size [nm]
Biological Activity/
Effect/Outcome
Tested Organism
-
ZnO
ZnO
ZnO
Zn(NO3 )2
Zn(O2 CCH3 )2
Zn(O2 CCH3 )2 ·(H2 O)2
Aqueous leaf extract of Solanum nigrum
Leaf powder aqueous extract of
Scadoxus multiflorus
Leaf extract Atalantia monophylla
Ref.
The higher antimicrobial activity was found against
S. paratyphi compared to the standard tablet;
less bactericidal activity was found for S. aureus, V. cholera and
E. coli than a standard tablet.
The leaf extract also showed antimicrobial activity against
V. cholera and S. aureus.
[178]
S. aureus, Salmonella
paratyphi, Vibrio cholerae,
E. coli
-
31 ± 2
Aedes aegypti (larvae and
eggs); Aspergillus niger;
Aspergillus flavus
-
Less larvicidal activity when compared to the literature;
96.4% at 120 ppm ovicidal activity of the mosquitos;
small fungicidal activity against A. niger and A. flavus.
[179]
-
20–45
Bacterial (B. subtilis,
B. cereus, S. aureus,
P. aeruginosa,
Klebsiella pneumonia) and
fungal species (C. albicans,
A. niger)
The biosynthesized ZnO NPs were found to outsmart
conventional antibiotics and plant extracts in the destruction of
pathogenic microorganisms.
Bacterial strains had a high susceptibility to ZnO NPs when
compared to fungi.
[180]
The better antibacterial activity of ZnO NPs (100 mg mL−1 )
than standard antibiotic (gentamycin—100 mg mL−1 );
Better biocidal activity than other researchers findings and
low MIC.
[181]
-
The zone of inhibition was 4.8 mm at 90 ◦ C and 4.3 mm at
room temperature for ZnO NPs.
[182]
-
The higher bactericidal effect against gram-negative
than gram-positive.
[183]
-
Low concentrations of ZnO NPs were required for the
complete prevention of growth of these organisms in vitro.
[184]
20–30
ZnO
Zn(O2 CCH3 )2 ·(H2 O)2
Green tea leaves (Camellia sinensis)
30–40
G+ (S. aureus) and G−
(E. coli) bacteria; fungal
species (A. niger)
ZnO
Zn(O2 CCH3 )2 ·(H2 O)2
Aqueous extract of parsley
(Petroselinum crispum)
50 nm (at RT)
40 nm (at 90 ◦ C)
E. coli
ZnO
ZnSO4
Leaf extract of Bauhinia tomentosa
22–94
G− (P. aeruginosa, E. coli)
and G+ (B. subtilis,
S. aureus)
ZnO
Zn(O2 CCH3 )2
Leaf extract from Stevia
10–90
Parasitic strain:
Leishmaniasis major
Bacteria: S. aureus and
Escherichia coli
-
-
-
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Table 1. Cont.
MO
Precursor
Biosynthesis
NP Size [nm]
Tested Organism
ZnO—16.7;
Cu-doped ZnO
method 1–17.5;
Cu-doped ZnO
method 2–20.7
Bacteria: S. aureus,
B. subtilis, Klebsiella,
E. coli; fungal strains
A. niger, A. flavus,
Trichoderma harzianum;
An anticancer activity
using human breast
carcinoma cells
Biological Activity/
Effect/Outcome
-
ZnO and Cudoped ZnO
RuO2
CeO2
NiO
Zn(NO3 )2 ·6H2 O and
Cu(NO3 )2
RuCl3 ·xH2 O
CeCl3
NiO(CH3 COO)2 ·4H2 O
1.
2.
Aqueous leaf extract of Abutilon indicum
(for ZnO NPs)
Extracts of Clerodendrum infortunatum
(1 for Cu-doped ZnO NPs)
Clerodendrum inerme (2 for Cu-doped
ZnO NPs);
Plant extract of Acalypha indica
Leaf extract of Gloriosa superba L.
Citrus fruit juice of
Limoinia acidissima Chrism
6–25
E. coli,
P. aeruginosa,
Serratia marcescens
S. aureus
5
E. coli, S. aureus,
S. dysenteriae,
P. aeruginosa, P. vulgaris,
K. pneumonia,
S. pneumoniae
20
G+ (S. aureus) and G−
(P. aeruginosa, E. coli,
K. pneumonia) bacteria
-
-
-
-
-
-
Ref.
ZnO NPs and Cu-dopped ZnO (method 1) showed effective
antibacterial activity against Klebsiella and B. subtilis;
Cu-dopped ZnO (method 2) indicated superior and very
effective broad-spectrum antibacterial activities because of
higher Cu-doping and larger surface area;
Antifungal potential against A. niger and T. harzanium was
observed more in method 2 MONPs than in method 1;
Cu-doped ZnO from method 1 and 2 showed a higher death
rate for cancer, in contrast to control (standard drug).
[185]
High antibacterial activity against both gram-negative (E. coli,
P. aeruginosa and Serratia marcescens) and gram-positive
(S. aureus) bacteria.
[186]
At 100 mg CeO2 NPs showed the most significant effect on the
zone inhibition of S. aureus. P. aeruginosa, P. vulgaris,
K. pneumonia, S. pneumonia and E. coli showed similar
inhibition zone with 50 mg NPs;
Gram-positive bacteria are relatively more susceptible to NPs
than gram-negative.
[187]
The highest was the bactericidal activity against E. coli
followed by S. aureus;
Less antibacterial activity was recorded against K. pneumonia
and P. aeruginosa.
[188]
Biosynthesis of MONPs from bacteria, fungi, algae and natural compounds
ZnO
ZnO powder
Culture of bacteria Aeromonas hydrophila
42–64
Aeromonas hydrophila,
E. coli, S. aureus,
P. aeruginosa,
Enterococcus faecalis,
Streptococcus pyogenes;
Aspergillus flavus, A. niger,
C. albicans
-
The maximum zone of inhibition was observed in ZnO NPs
against P. aeroninosa and A. flavus;
A. hydrophila, E. coli, E. feacalis and C. albicans showed
minimum inhibition concentration at 1.2, 1.2, 1.4
and 0.9 µg mL−1 for the MONPs.
[189]
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Table 1. Cont.
MO
Precursor
Biosynthesis
NP Size [nm]
Biological Activity/
Effect/Outcome
Tested Organism
-
ZnO NPs exhibit high biocompatibility against hMSC and
proved to be potentially safe in mammalian cells;
Anti-H. pylori dosage of ZnO NPs was safe to human
mesenchymal stem cells (hMSC) and could effectively be used
as a nano-antibiotic.
[190]
The MIC of TiO2 NPs was 40 µg mL−1 for S. aureus,
40 µg mL−1 for E. coli, 80 µg mL−1 for P. aeruginosa, 70 µg mL−1
for K. pneumoniae and 45 µg mL−1 for B. subtilis.
[191]
The effects of ZnO NPs against gram-positive were higher than
that against gram-negative bacteria;
The inhibitory effect increased with concentration;
The antibacterial activity of ZnO NPs was comparable to that
of conventional antibiotic ciprofloxacin (0.5 mg mL−1 ).
[192]
-
Impregnated fabrics with ZnO NPs showed zones of inhibition
around 12 and 10 mm against S. aureus and E. coli, respectively.
[193]
-
The radial diameters of the inhibition zone of E. aerogenes and
S. aureus were 14 and 16, respectively, therefore, gram-negative
seemed to be more resistant to CuO NPs than
gram-positive bacteria;
Significant antibacterial activity against both gram
classes bacteria.
[194]
The largest radial diameter of inhibition for gram-negative
bacteria was in P. vularis (around 28 mm);
The maximum inhibitory effect against gram-positive was
found in B. cereus (around 27 mm).
[195]
ZnO
Zn(NO3 )2
Culture of Bacillus megaterium
45–150
Helicobacter pylori
-
TiO(OH)2
Mycelium of Aspergillus flavus
62–74
S. aureus, E. coli,
P. aeruginosa,
Klebsiella pneumoniae,
B. subtilis
-
TiO2
ZnO
ZnCl2
A fungal isolate of Aspergillus niger
41–75
S. aureus, E. coli
ZnO
Zn(NO3 )2
Culture medium of Aspergillus niger
84–91
S. aureus, E. coli
CuO and
Cu2 O
CuO
CuSO4
Cu(O2 CCH3 )2 ·H2 O
Algae extract of brown algae
Bufurcaria bufurcata
Algae extract of cyanobacteria
Spirulina Platensys
5–45
30–40
Enterobacter aerogenes,
S. aureus
G−: E. coli,
Proteus vulgaris,
Klebsiella pneumonia
G+: S. aureus;
S. epidermidis,
Bacillus cereus
Ref.
-
-
-
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Table 1. Cont.
MO
Precursor
Biosynthesis
NP Size [nm]
Tested Organism
4–23
S. aureus—sensitive
and resistant;
C. albicans—sensitive
and resistant;
Biological Activity/
Effect/Outcome
-
ZnO
Zn(NO3 )2 ·6H2 O
Algal extract of marine microalgae
Sargassum multicum
-
ZnO
ZnNO3 ·6H2 O
Al-gum extrudates of Azadirachta indica
30–60
E. coli, S. aureus
CuO
CuSO4
Goat (GFM) and sheep (SFM) fecal matter
29.2 ± 15.9
for GFM;
32.3 ± 32.2
for SFM
Salmonella typhi, B. subtilis
-
-
Ref.
The inhibition zone in both sensitive and resistant strains
was comparable;
The zones of inhibition were about 28 and 25 mm for
C. albicans and S. aureus, respectively;
The bactericidal activity of cotton fabrics impregnated with
ZnO NPs was reduced by 2–25% after washing.
[196]
All tested bacteria showed resistance to ZnO NPs synthesized
by the green method as compared with bulk ZnO;
At concentration 10 µg mL−1 , both gram-positive and
gram-negative bacteria indicated good sensitivity.
[197]
CuO (GFM and SFM) NPs demonstrated significant
antimicrobial activity against both bacteria compared
to ampicillin.
[198]
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3.8.1. Titanium Dioxide Nanoparticles
TiO2 in the anatase phase is shown to be the most potent form of producing ROS [199]. According to
Cho et al. [200], superoxide alone played an insignificant role in the inactivation mechanism of bacteria
in contrast to OH• radical which was primarily responsible for E. coli inactivation. The photooxidation
decomposition and mineralization peroxidation of the bacterial plasma membrane saturated and
unsaturated fatty acids are thought to be one of the reasons for high antibacterial activity [201].
Another study confirms that TiO2 NPs reacted with thiol (-SH) groups of the proteins in bacterial
cell wall causing inactivation of transport proteins nutrients and reduction of cell permeability which
triggered cell death [202]. Moreover, the bactericidal activity of UV-light-activated anatase TiO2 NPs
was dependent on the amount of dissolved molecular oxygen and the proper cell-NPs contact that
accelerated the translocation of NPs across the microbe cell wall [203]. TiO2 NPs photocatalysis showed
different dysfunctions such as cell inactivation at the regulatory and signaling level, a decrease in
coenzyme–independent respiratory chain, lower ability to transport and assimilate Fe and P, and lower
capacity of heme biosynthesis and degradation [204]. With the addition of metals such as Pt, Au,
Ag, Ni, and Cu to TiO2 NPs, the UV-light-activated bactericidal activity of nanocomposites against
E. coli was found to be greater compared to pure TiO2 NPs [205]. Simultaneously, in the absence
of photoactivation, TiO2 NPs were not losing their antimicrobial activity that could be explained
with the electromagnetic attraction between the microorganisms and MONPs that induced oxidation
reactions [206]. The antimicrobial activity of TiO2 NPs modified with G. zeylanica (endemic plant of
Sri Lanka) aqueous extract was enhanced because of the multiple mechanisms of phytochemicals
and when exposed to sunlight, the bactericidal activities of the modified TiO2 NPs were additionally
improved [207]. Titania NPs showed inhibitory activity towards H9N2 avian influenza virus which
activity increased under UV light [208]. TiO2 NPs tagged with DNA encoding region of the viral DNA
was able to inhibit H5N1 and H1N1 viruses replication [209].
3.8.2. Zinc Oxide Nanoparticles
ZnO NPs hold high optical absorption in UVA and UVB spectrum which property is important
for their antibacterial response and protection of UV light in cosmetics. The bactericidal effect
of ZnO against gram-negative [210] and gram-positive [211] bacteria was found to be dependent
on the oxide concentration and size of MONPs [212]. As an n-type semiconductor, ZnO NPs in
aqueous solution absorbed UV irradiation and showed phototoxic effect producing ROS like H2 O2
and superoxide ions (O2 •− ) that could inhibit or kill microorganisms [213] by interacting with active
enzymes, proteins, and DNA [214]. It is established that high atom density (111) facets of ZnO
crystal lattice exhibited higher bactericidal activity [215]. The comparison of the antibacterial activity
of ZnO NPs in dependence on the size of particles indicated the best bactericidal response of the
particles with a smaller size [26] because of the concentration of oxygen species on the surface was
higher for the higher surface area. TEM analysis of S. aureus and B. cereus membranes showed
that ZnO NPs caused bacteria cell deformation and damaging together with disorganization of the
intercellular structures [216]. Some authors determined that the antibacterial activity did not correlate
with ROS levels or Zn2+ ion release [217] while others explained the damage of cell membranes with
creating electrostatic forces between the negatively charged cell surfaces and ZnO NPs containing
positive charges in water suspension [218]. ZnO NPs mediated killing of S. aureus caused significant
up-regulation of pyrimidine biosynthesis and carbohydrate degradation while the amino acid synthesis
was down-regulated [219]. ZnO NPs showed not only antibacterial but also antifungal activity
against A. invadans at a concentration lower than that of silver NPs against the same fungus while
simultaneously caused higher cytotoxicity [220]. Because of the attraction of negatively charged ZnO
NPs towards viruses, ZnO tetrapod type structures were able to control and trap both herpes simplex
virus HSV-1 and HSV-2 [221,222] which make them of specific interest in prophylactic therapy and/or
maintaining latency.
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3.8.3. Copper Oxide Nanoparticles
CuO NPs are far cheaper than AgO NPs but higher concentrations are required to obtain the
anticipated antimicrobial effect. It is believed that the microbial toxicity of CuO NPs is mainly due
to Cu2+ ion release [223,224]. CuO NPs are more effective towards bacterial species with cell walls
rich in amine and carboxyl groups such as gram-positive B. subtilis [225]. Since the multilayered
peptidoglycans in gram-positive strains are negatively charged, they can bind Cu2+ ions released from
CuO NPs. At pH 6 and 7, gram-positive S. aureus strains were more resistant to CuO NPs as opposed to
pH 5 where the higher toxicity of CuO NPs was related to increased release of Cu2+ and induced ROS
response [226]. The smaller CuO NPs with a size of around 20 nm appeared to have strong bactericidal
activity against both gram-positive and gram-negative bacteria [227]. Concerning gram-negative
E. coli, Cu2 O (cubic) NPs were more efficient against the microorganisms than CuO (monoclinic)
showing higher affinity to the bacterial cell while CuO NPs produced significant ROS in terms of
superoxides than Cu2 O [228]. At concentration 50 µg mL−1 , CuO NPs had higher biocidal activity
against microorganisms of oral microbiota than ZnO NPs [229] without showing genotoxic or cytotoxic
effects on cancerous HeLa cells in the same doses [230]. CuO NPs did not have M13 bacteriophage
inactivation activity, whereas dual photoexcitation by UV irradiation-induced not only bactericidal but
significant anti-phage activity [231]. Besides, the multiple responses of P. aeruginosa exposed to CuO
NPs induced lysogenic bacteriophage which might render defective within the bacterial host such as
nitride accumulation increased N2 O emission and inhibited respiration [232].
3.8.4. Silver Oxide Nanoparticles
Silver has a beneficial effect to be toxic even at low concentrations against bacteria [233]. It is
generally accepted that highly reactive silver ions lyse or ultimately kill the bacteria cells. Similarly, silver
is known to interact with the thiol groups (-SH) of proteins or generate ROS which contributes to the
antimicrobial properties [204–234]. The accumulation of silver NPs in microbial membranes was shown
to cause increased permeability and finally, the membrane was damaged by the free radicals formed
by the presence of NPs [235]. The bactericidal activity of AgO NPs produced by microbial cultures
was almost the same as that of silver NPs and they were effective against both gram-positive cocci
and rods [236]. The hemolysis effect of Ag2 O NPs prepared either by chemical or green methods with
similar sizes (~30 nm) and almost the same spherical shape was found to be highly different [237] from
Ag NPs that were stable in nature and had lower hemolysis effect. The oxide NPs showed an increase
in lysis properties due to redox processes triggering interfacial charge interactions. The comparison
of functionalized with 5-amino-2-mercapto benzimidazole Ag2 O3 NPs with non-functionalized,
demonstrated that antimicrobial activity against S. aureus and P. aeruginosa of the functionalized NPs
was decreased [238]. Additionally, Ag2 O3 NPs demonstrated significantly toxic effects against the
fungus A. niger in contrast to the modified NPs. The 5-amino-2-mercaptobenzimidazole did not
have antibacterial behavior against gram-positive but possessed excellent bactericidal effect against
gram-negative. The authors explained the reduced bactericidal activity of the functionalized NPs with
the reduced interaction of Ag2 O3 NPs with the cell membrane and a slight increase in particle size.
3.8.5. Magnesium Oxide Nanoparticles
MgO NPs trigger post activation of the bone-repair scaffold and are used as hyperthermia agents
in cancer therapy [239]. MgO NPs exhibited effective bactericidal potential against both gram-positive
(S. aureus) and gram-negative (E. coli) bacteria [240] and even against fungi such as Aspergillus niger and
Penicillium oxalicum [241]. MgO NPs demonstrated better inhibition for rod-shaped bacteria (E. coli)
in contrast to spherical-shaped bacteria [242]. Portions of MgO NPs possibly react with water to
form Mg(OH)2 which could lose OH− ion into the solutionand increase the pH. It was previously
demonstrated that the peptide linkage in the cell membrane of Pseudomonas aeruginosa and E. coli
was destroyed by the generated superoxide ions on the surface of MgO NPs [243]. O2 − was more
Biomimetics 2020, 5, 27
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stable in the alkaline environment that contributed to the higher antibacterial effect of MgO NPs [244].
The mean zeta-potential of MgO NPs exhibits a positive charge in pH range from 4 to 8 [245]
favoring the electrostatic interaction of NPs with bacteria cells. These MgO NPs could completely
kill phytopathogen bacteria R. solanacearum at a comparatively higher concentration, 250 µg mL−1 .
Similarly to the other MONPs, MgO NPs destroyed and disintegrated the cell wall of the phytopathogen
bacteria leading to leakage of the intercellular content and cell death. The same authors discovered that
except for retained biofilm formation, MgO NPs improved the bacterial susceptibility to antibiotics.
In contrast to nisin (antibiotic) addition, MgO and ZrO mixing did not enhance MgO activity against
pathogens [246]. MgO NPs possess also the advantage of not being cytotoxic to human cells at lower
concentrations (0.3 mg mL−1 ) [247]. However, the bactericidal activity of MgO NPs increases with
raising the concentration of NPs.
3.8.6. Calcium Oxide Nanoparticles
Similarly to MgO NPs, the formation of ROS in the presence of CaO NPs was influenced by the
higher pH that helped in antibacterial activity [248]. For both MgO and CaO, the contact between
NPs and bacteria is an important factor for the bactericidal activity [249]. The active superoxides ions
reacted with the carbonyl group in the peptide linkages of the cell wall of bacteria and disrupted
them [250]. High-concentrated CaO and MgO NPs slurries in physiological saline were even able to
kill spores of B. subtilis that are more robust with their thick proteinous wall than the vegetative cells.
In contrast to the little affinity of ZnO NPs to fungal cells, the alkali MONPs – CaO and MgO, showed
stronger affinity to postharvest fungal cells (Saccharomyces cerevisiae, Candida albicans, Aspergillus niger
and Rhizopus stolonifer) than to bacterial cells [251]. This suggests that the yeasts were killed by the
high pH of the alkali oxides because of the solubilization of cell surface proteins and the presence of
alkali-soluble polysaccharides [252].
3.8.7. Aluminum Oxide Nanoparticles
It was found that alumina NPs worked against E. coli only at high concentrations [253]. Al2 O3 NPs
synthesized by co-precipitation method with irregular shape and size of around 35 nm demonstrated
effective antibacterial activity not only against E. coli but also against P. vulgaris, S. aureus and
S. mutans [254]. The highest was the susceptibility of E. coli and the smallest for P. vulgaris. Like the
other oxide NPs, their interaction with the cell wall distorts the cell morphology. Ansari at al. [253]
characterized the damage of the E. coli cell wall under the influence of Al2 O3 NPs as the formation
of pits. Agglomerated particles remained adherent to the outer surface while small-sized NPs were
found uniformly distributed inside the cells. The lipopolysaccharides from the cell wall-bound to
alumina NPs in a similar manner as the bacterial cell bound to a solid surface. It was proposed that the
interaction between the amphiphilic biomolecules and Al2 O3 NPs could affect the membrane fluidity
and integrity explaining the antibiosis of NPs. Jiang et al. [255] examined whether the bactericidal
activity of alumina NPs was due to the particles in the suspension or due to the ion release which could
damage the cell membrane. It was established that there were no dissolved aluminum ions detected in
the supernatant of suspension leading to the conclusion that the attachment of NPs to the cell wall
was of prime importance for the antibacterial activity of alumina NPs. Alumina-silver composite NPs
produced by the wet chemical method and modified with oleic acid as a capping agent demonstrated
slightly higher antimicrobial activity against gram-positive S. epidermidis bacteria than against E. coli
but both bacteria were more sensitive to the complex NPs as opposed to alumina NPs themselves [256].
3.8.8. Iron Oxide Nanoparticles
The magnetite (Fe3 O4 ) and maghemite (Fe2 O3 ) indicate single crystalline in structure biocompatible
materials showing superparamagnetic behavior. Due to the strong magnetic attraction among NPs
and high energy surfaces, they tend to agglomerate. Combining Fe2 O3 NPs with Ag NPs could avoid
coalescence and contamination of the surrounding by achieving the recycling of nanosized silver [257].
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The resultant heterodimer NPs base on magnetic core coated with ethylenediamine ((CH2 )2 (NH2 )2 )
modified chitosan/polyacrylic acid (PAA) and Ag NPs demonstrated excellent antibacterial against
both S. aureus and E.coli because of the electrostatic sorption to bacteria cell and the enhanced efficiency
by Ag NPs. The iron oxide–magnetite, is of great interest because of high magnetism, biocompatibility,
low cost and low toxicity [258]. Fe3 O4 NPs synthesized by top-down pulse laser ablation in two
types of solutions, SDS and DMF (dimethylformamide) indicated high biocidal action against both
gram-positive (G+) and gram-negative (G−) bacteria [259]. The stress in G− bacteria the authors
explained with the formation of ROS generated with the interplay of oxygen with reduced iron species
(Fe3+/ Fe2+ ) or disturbed electronic or ionic transport chains in the cell membrane. The higher Fe2 O3
NPs effectiveness against G+ bacteria the authors related to the smaller negative charge of S. aureus
membrane than that of E. coli causing a higher level of penetration of the negatively charged NPs.
The high antimicrobial activity of iron oxide NPs synthesized using leaf extract of Cynomtra ramiflora
against E. coli and especially against S. epidermidis attributed to the synergetic effect of the phytochemical
residing in the leaf extract and ROS produced by NPs [260]. These results suggested a synergetic effect
of the phytochemical alkaloids and generation of ROS by the oxide NPs.
3.8.9. Nickel Oxide Nanoparticles
NiO NPs were found to have bactericidal and bacteriostatic activity depending on the bacterial
species and concentration. We synthesized Nickel-based Gadolinia doped ceria with spherical shape
and size between 40–70 nm with composition NiO-Ce0.8 Gd0.2 O2-δ via co-precipitation [261]. At a
concentration equal to 100 µg mL−1 , the antibacterial activity of the prepared NiO-CGO nanocomposite
against Gram-negative (E. coli and V. cholerae) strains was found to be slightly lower than that of
standard antibiotic (Streptomycin). After surface functionalization, both the bactericidal activity
(against S. aureus and P. aeruginosa) and antifungal activity of 5-amino-2-mercaptobenzimidazole NiO
NPs were increased compared with the non-functionalized NiO NPs [262]. The authors explained
the observed difference with the enhanced dispersibility of the modified NPs. As Ni ions could not
pass through the cell membrane ion channels, small NPs must have been uptaken by the cells and
extracellular Ni ions might interfere with intracellular Ca2+ metabolism, which might be the reason for
cellular damage. NiO NPs are also shown to interact with the sulfur and phosphorous of the bacteria
DNA and other functional groups of proteins, which led to protein leakage and bacteria death [263].
Green synthesized NiO NPs annealed at different temperatures showed moderate bactericidal activity
toward suspension of microbial pathogens S. aureus and E. coli without completely inhibiting the cell
growth of both strains [264].
3.8.10. Cerium Dioxide Nanoparticles
In contrast to other NPs, CeO2 inhibits ROS rate of production, induced by H2 O2 in a
dose-dependent manner that suggests their high anti-oxidant potent [37]. CeO2 NPs showed activity
against both the gram-positive and gram-negative bacteria but the greatest antimicrobial function was
observed against gram-negative strains [265]. This is because CeO2 NPs are positively charged and
easily bind to gram-negative bacteria by electrostatic attraction [266]. In contrast to other NPs, CeO2 NPs
were not found to penetrate through the E. coli membrane [267]. The proposed mechanisms for bacteria
inactivation were: electrostatic adsorption, modifying the cellular ion transport; oxi-reduction processes
on the surface of NPs, resulting in oxidative stress on lipids and proteins of the plasma membrane;
an attack on the electron-flow and bacterial respiration. CeO2 NPs synthesized by employing a
solution combustion method with ceric ammonium nitrate and EDTA as fuel at 450 ◦ C, demonstrated
spherical form with an average size of 42 nm [268]. The authors found concentration-dependent
bactericidal activity against P. aeruginosa and a lack of activity against gram-positive S. aureus. It was
proposed that the reason for the observed effect was related to the presence of teichoic acid in the
peptidoglycan layer of gram-negative bacteria that interact with CeO2 NPs. Recent examinations focus
on the antimicrobial activity of Zr doped CeO2 NPs with different concentrations of Zr synthesized by
Biomimetics 2020, 5, 27
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precipitation method [269]. The varying ratio of Zr impregnated CeO2 NPs (5 to 15%) did not greatly
influence the moderate antimicrobial activity against S. aureus, E. coli, P. aeruginosa, S. faecalis, B. subtilis
and P. vulgaris of the modified NPs. It was also found out that these impregnated with Zr CeO2 NPs
were not effective against pathogenic fungi like C. albicans, A. terreus, C. tropicalis, A. flavus, A. fumigatus
and A. niger.
4. Nanotoxicology
Nanotechnology constitutes a very encouraging field for the production of new diagnostics and
therapeutic agents with applications in medicine but merely a few nanoproducts were currently used
in practice. The oxide NPs show good potential for therapy of different diseases and pathogen control.
Besides the important biological functions of ROS such as cell proliferation, migration, signalization,
wound healing and so forth, the generated by MONPs reactive species could produce not only an
inflammatory response but generate oxidative stress above a certain amount and eventual apoptosis of
normal cells. For example, Rizk et al. [47] demonstrated that the intoxication with TiO2 NPs caused
complex multifactorial ailment routes, leading to chromosomal aberrations in bone marrow metaphase
cells of intoxicated mice and surge in the activity of serum aspartate and alanine aminotransferase
which is the markers designating severe inflammation and liver injury as a result of oxidative stress,
relative to size and composition of NPs. Most of the researchers face with the issue of balance amongst
the therapeutic or diagnostic effects of MONPs and their toxic side effects. The toxic effects are greatly
dependent on the physicochemical properties of NPs, concentration, time of interaction, NPs’ stability
in a certain environment, type of the metal oxides and cells tested, and MONPs’ accumulation in tissue
and organs.
It now becomes clear that it is difficult to distinguish NPs-specific effects from the ionic effect
that usually occur simultaneously. The ion dissolution is influenced by the type of NPs, their pH,
ionic strength and interfering organic matter. Additional complexity is found when NPs tend to
agglomerate. The low colloidal stability results in a decrease of NP concentration in the active
media. Internalized in the body, NP aggregates were not broken up by biofluids and remained
intact during distribution [270]. Moreover, NP aggregation that is affected by pH, temperature,
ionic strength and so forth could change the behavior, mobility, bioavailability, and, therefore,
the fate of the NPs in the organism. The interaction between NPs and macromolecules, cells, tissues
and organs is critical in determining their behavior in biological systems. It has been postulated
that the cell surface-NPs charge interaction enhanced the metabolic activity of the cells [271] but
when the concentration of NPs increased to 300 µg mL−1 both proliferation and differentiation are
hindered probably because the internalized NPs induce apoptosis [64]. NPs such as γ-Fe2 O3 with no
dose-dependent action demonstrated in vivo toxicity in rats due to biotic and abiotic dynamics and
lack of target delivery. Moreover, 10 nm poly(ethylenimine)-coated iron oxide demonstrated in vivo
dose-dependent lethal toxicity in Balb/c mice [12]. When examining the viability of mouse embryonic
fibroblasts NIH3T3, Jarockyte et al [272] found that after 48 h of testing a dose-dependent decrease in
cell viability is observed and only a low concentration of iron oxide NPs (32.5 ng mL−1 ) was completely
eliminated within three weeks. Multicore iron oxide nanoflowers of around 140 nm administrated
to a Xenopus Laevis animal model indicated that although the particles were not lethal in elevated
doses, several malformations were observed in the embryos [273]. The genotoxicity and hematological
changes of long-term (60 days) and low-dose (100 and 200 mg kg−1 b wt) oral exposure to anatase
TiO2 NPs (5-10 nm) manifested swollen erythrocytes and macrocytic anemia while activation of the
defense and immune system and inflammation occurred in the lungs, kidney, liver, and brain [274].
At the cell level, the effect of TiO2 NPs on human neuroblast cells (SH-SY5Y) indicated a lesser
growth rate, greater shortening rate of microtubules, and condensed lifetimes of de novo synthesized
microtubules [36–51]. In an interesting study, the toxicity of TiO2 nanowhiskers in combination with
5,10,15,20-tetra(sulfonatophenil)porphyrin (TSPP) assessed in vivo on rat was reduced as compared to
pure TSPP especially at high concentrations [275]. According to MTT assay, the combination of 0.1 mM
Biomimetics 2020, 5, 27
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TSPP with 0.6 mM TiO2 revealed minimized cytotoxic effects because of the porous nature of NPs
allowing a slow release of the adsorbed TSPP that helped in lowering the adverse effects.
Although Zn can be efficiently excreted from the body through urine, sweat, and feces, ZnO seems
to cause an inflammatory response and in vivo toxicity [276]. When instilled into the respiratory tract,
ZnO NPs induced lung inflammation by the production of pro-inflammatory cytokines via Toll-like
receptor (TLR) signaling pathways in a myeloid differentiation primary response protein-88 dependent
manner [277]. The intravenous injection of 2.4 mg kg−1 ZnO nanorods in mice model caused reduced
platelet and blood cell counts, increased stress markers in the liver together with DNA damage in
the spleen, liver, and kidney [278]. Injected at higher doses (30 mg kg−1 ) in rats, ZnO NPs triggered
multifocal acute injuries in the lungs and elevated mitotic figures in hepatocytes [279]. Han et al. [280]
reveal that the daily intraperitoneal injection of ZnO NPs to young Wistar rats over 8 weeks affected
the functionality of the brain by enhancing the long-term potentiation while scarcely influencing the
depotentiation in the dentate gyrus region of the hippocampus. This bidirectional effect of ZnO NPs on
long-term synaptic plasticity broke the balance between stability and flexibility of cognition which could
modulate the efficiency of spatial cognition. Consequently, before implementing toxicity assessment
the particle size, distribution, composition, morphology, surface area and chemistry, intrinsic properties
and particle reactivity in the solution should be taken into consideration for accurate characterization.
If the adoption and succeeding distribution are highly irrelevant, the impending risk upon exposure
can be limited to local and indirect effects, while the sluggish elimination or whole retention result in
NPs buildup and increased internal exposure [270].
Another important parameter influencing the long term toxicity of MONPs is their biodegradation.
Highly acidic media with certain proteins found in lysosomes can degrade the particle coatings
and trigger NP corrosion [281]. By incubation for 24 h with artificial lysosomal fluid (pH 4.5),
ZnO NPs dissolved around 90%, whereas, in artificial interstitial fluid (pH 7.4), the particles showed
no dissolution [282]. The elevated Zn2+ ion concentration increased intercellular ROS, DNA damage
and triggered apoptotic cell death [283]. In the degradation of nanocube iron oxide NPs, the formation
of ferritin in a murine model was observed proving the activation of iron metabolism routes that deal
with iron coming from NP degradation [284]. The presence of coating and aggregation of NPs are
critical parameters decreasing the degradation kinetics. Recently, it was described that in tumor cells,
transitional metals like iron, copper and so forth contribute to regulated nonapoptotic cell death called
ferroptosis brought by oxidative stress response and presenting no morphological or biochemical
markers of cell death [285]. Ferroptosis exhibited many similarities to cell death called oxytosis such as
the disintegration of intercellular organelles, glutathione depletion with loss of glutathione peroxidase
4 activity, ROS generation and an increase of cellular calcium. The reduced capacity of iron storage in
serum ferritin complexes on one hand and on the other - the increase in iron uptake through either
the membrane-bound transferrin receptor 1 (ThR1) or within endosome compartments, contribute to
intercellular iron overload and subsequent generation of highly reactive hydroxyl radicals creating
lipid hydroperoxides and production of lipid ROS [286]. Mitochondria, lysosomes, endoplasmic
reticulum and cell membrane contributed to overall levels of ROS production and together with the
increased cytoplasmic calcium, they could eventually trigger oxidative cell death. It follows that when
NPs start degrading ferroptosis will have a negative effect but if new oxide-based NP constructs with
controlled atom release are designed, they can be used as a positive tool in cancer therapy.
Following the tabulated studies, it is extremely hard to draw an unambiguous conclusion regarding
the relevance of ion release and ROS from NPs to the various toxicological effects of the nanosized
agents. NPs of the same chemical composition occurring in different nanoforms concerning size,
shape, surface affinity and so forth, may exhibit different hazard and fate profiles [287]. Add to
this, the combined exposure of MONPs with other chemical substances can trigger unexpected and
contradictory results. Moreover, the impact of the coating or modifying shell is not a matter of
indifference. The relative biological effects of NPs with different initial characteristics such as chemistry,
size, shape and crystallinity which characteristics usually trigger mixture toxicity effects (such as
Biomimetics 2020, 5, 27
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protein binding, enzyme activation, different NP interactions, etc), induce different sensitivity while
using different cytotoxic assays which may lead to diverging or wrong conclusions. Therefore, some of
the common methods have to be upgraded and adapted and new ones together with test guidelines
and regulatory frameworks for in vivo, in vitro, and/or in silico testing of NP toxicological effects have
to be developed.
5. Summary and Future Prospectives
The primary goal of this review was to set a reference to researchers who are interested in
nanoparticle-based functional biomedical applications. MONPs possess huge potential as substances
fighting multidrug-resistant microorganisms that could substitute antibiotics. The redox-active MONPs
tend to modulate the human innate and adaptive immunity and this ability could be successfully
used in enhancing immune response during vaccination or mediating immune tolerance against
autoimmune diseases, allergies or cancer. The application of MONPs as molecular imaging agents,
drug vehicles, and cancer therapeutics is extremely promising but there are many challenges and
emerging problems that need to be solved before clinical or industrial applications. Despite the huge
potential of MONPs in the biomedical area, the major drawback of ROS-based therapy is the inability
to control ROS delivery to tissues or cells. However, there are also certain unknown mechanisms of
cellular and extracellular functioning of MONPs that are unrelated to ROS generation waiting to be
successfully established.
MONPs were debated in detail for their application in nanomedicine as tissue and
immunotherapeutics, quantum dots, in dentistry, regenerative medicine, wound healing, also as
biosensors. Their antimicrobial, antifungal and antiviral properties were also well discussed. With a
vast number of different metal oxide NPs, only the majority of examples could be mentioned here by
emphasizing the principal advantages of such MONPs. MONPs have emerged as a new generation
of materials that go further than conventional applications of bulk materials. The variety of unique
properties makes them potentially of great use in fuel cells, energy storage and conversion, catalysis,
solar cells, optical displays, sensors and many other electronic and optoelectronic devices [6].
Considering certain applications, the biological effects and cytotoxicity should be conferred
concerning MONPs type, size, concentration, synthesis method, and so forth. This should also include
in vivo examinations of the long-term health risks. Both advantages and disadvantages represent
a challenge sharing material scientists and life scientists who ought to develop and examine the
structure-biological performance interrelation of particular therapeutic MONPs. The development
of functional, biocompatible and safe MONPs used in nanomedicine treatment of human diseases
should be based on the proper understanding of all interactions between regulatory and activating
factors and toxicity mechanisms. If the molecular regulation mechanisms of redox- and other signaling
pathways that are triggered by NPs are profoundly understood then improved control over the cell
cycle, migration, proliferation, differentiation, angiogenesis and so forth, could be exercised.
Besides all disadvantages, a new avenue for chemotherapy, immunotherapy, bone and wound
healing and so forth has been provided by the development of NPs. The continuous advances in
the development of engineered MONPs supplement multimodal approaches based on new and
sophisticated methods for therapy and diagnosis of pathologies. Devices of metal oxide NPs and
nanotechnologies have proven to be beneficial not only in smart drug delivery but also as theranostic
platforms of non-invasive imaging. Since current diagnostic technologies face difficulties in the
fading of fluorescent dyes due to bleeding, fluorescent NPs can overcome these shortcomings [288].
Metal oxide nanomaterials make it feasible to achieve smart drug delivery systems for lethal diseases
that demand site-specific treatment with minimal side effects. Researches in cancer therapy are
vigorously seeking multi-therapy options and remote control of the functions of NPs. Since molecular
profiling of tumors and other diseases is unique, the selection of targeting agents or treatment based on
the molecular profile will pave an avenue for targeted personalized medicine with a highly appropriate
therapeutic strategy. This will open new doors to providing safer, more effective and customizable
Biomimetics 2020, 5, 27
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treatment options soon. This new area of individualized medicine will allow speeding up clinical trials
and health promotion of cancer patients.
Author Contributions: Authors M.P.N. and M.S.C. conceived of the presented idea and carried out a detailed
review. M.S.C. designed and supervised the whole work. In manuscript planning, articulation, the majority
writing part was done by M.P.N. with inputs and a substantial contribution from M.S.C. The authors equally
contributed to the work related to illustrations and tables. Also, both M.P.N. and M.S.C. thoroughly discussed the
contents, results and commented on the design of the figures, parameters in the tables and completed the final
manuscript review. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded by the National Science Fund of Bulgaria (NSFB), Contract № DN 07/3 (2016),
Gradient functional nanocoatings produced by vacuum technologies for biomedical applications.
Acknowledgments: M.S.C. acknowledges the research grants from the Department of Science and Technology
(DST), Government of India for the three major R&D projects: (a) SR/FTP/CS-116/2007, (b) No. SR/FT/CS134/2010
and (c) No. GITA/DST/TWN/P-002/2009 and Ministry of Science and Technology (MOST), Republic of China
(Taiwan) for its constant support through research grants, travel grants and many others.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to
publish the results.
Abbreviations
A549
AB
ADMA
Ag
APTES
ATP
Au
BBB
BC
BSA
CMX
CPR
Cr
DMF
DNA
FTL
G(-)
G(+)
HeLa
HFL1
HIV
HMSCs
IC50
IL-1α
IL-4
ITO
Ka
Kmapp
L-132
MCF-7
MO
MONPs
MRI
MTT
nm
NO
Adenocarcinomic human alveolar basal epithelial cells
Antibiotic
Exogenous asymmetric dimethylarginine
Silver
3-aminopropyl triethoxy saline
Adenosine triphosphate
Gold
Blood-brain barrier
Bacterial cellulose
Bovine serum albumin
Corona of the fusion protein
Cardiopulmonary resuscitation
Chromium
Dimethylformamide
Deoxyribonucleic acid
Ferritin light chain (a biomarker for ageing)
Gram-negative
Gram-positive
Henrietta Lacks (human cell line)
Human fetal lung fibroblast-1
Human immunodeficiency virus
Human mesenchymal stem cells
Half-maximal inhibitory concentration
Interleukin 1 alpha (hematopoietin)
Interleukin 4
Indium tin oxide
Association constant
Michaelis–Menten constant
Human lung epithelial cell
Michigan cancer foundation-7 (cancer cell line)
Metal oxide
Metal oxides nanoparticles
Magnetic resonance imaging
Methylthiazolyldiphenyl-tetrazolium bromide salt
nanometer
Nitrogen monoxide
Biomimetics 2020, 5, 27
NP
O2
PAMAM
PMMA-AA
PBS
PCL
PEG
PEI
pH
ppm
QDs
RNA
ROS
RT
SDS
-SH
SH-SY5Y
siRNA
SPAN
SPR
T-cell
THP-1
TNF-α
U mL-1
UV
VLP
33 of 47
Nanoparticle
Oxygen
Poly (amidoamine)
Polymethyl methacrylate–acrylic acid
Phosphate-buffered saline
Polycaprolactone
Polyethylene glycol
Polyethylenimine
Power of hydrogen
Parts per million
Quantum dots
Ribonucleic acid
Reactive oxygen species
Room temperature
Sodium dodecyl sulphate
Thiol groups
Human neuroblast cells
Small (or short) interfering RNA
Self-doped polyaniline nanofibers
Surface plasmon resonance
T-lymphocyte
Human monocytes
Tumor necrosis factor-α (inflammatory cytokines)
Units per millilitre
Ultra-violet
Virus-like particles
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