Transmucosal Nanoparticles:

Toxicological Overview 3
Swapnil Talkar, Sagar Dhoble,
Anuradha Majumdar, and Vandana Patravale

Abstract
Nanoparticles have specific physicochemical properties different to bulk
materials of the same composition and such properties make them very
attractive for commercial and medical applications. Mucoadhesive
nanoparticulate dosage forms are designed to enable prolonged retention
of these nanoparticles at the site of application, providing a controlled
drug release for improved therapeutic outcome. Moreover, drug delivery
across the mucosa bypasses the first-pass hepatic metabolism and avoids
the degradation by gastrointestinal enzymes. However, like most new
technologies, there is a rising debate concerning the possible transmucosal
side effects resulting from the use of particles at the nano level. In fact,
these nanoparticles on entering the body, deposit in several organs and
may cause adverse biological reactions by modifying the physiochemical
properties of living matter. Several investigators have found nanoparticles
responsible for toxicity in different organs. In addition, the toxicity of
nanoparticles also depends on whether they are persistent or cleared from
the different organs of entry and whether the host can raise an effective
response to sequester or dispose of the particles. In contrast to many efforts
aimed at exploiting desirable properties of nanoparticles for medicine,
there are limited attempts to evaluate potentially undesirable effects of
these particles when administered intentionally for medical purposes. This

S. Talkar · S. Dhoble · V. Patravale (*)

Department of Pharmaceutical Sciences and
Technology, Institute of Chemical Technology,
Mumbai, India A. Majumdar
e-mail: [email protected]; Department of Pharmacology and Toxicology,
http://www.vbpgroup-ict.in Bombay College of Pharmacy, Mumbai, India

© The Author(s) 2018 37

Q. Saquib et al. (eds.), Cellular and Molecular Toxicology of Nanoparticles, Advances
in Experimental Medicine and Biology 1048, https://doi.org/10.1007/978-3-319-72041-8_3
38 S. Talkar et al.

chapter focuses on the overview of the mucosal systems, fate of nanopar-

ticles, mechanism of nanoparticle’s toxicity and the various toxicity issues
associated with nanoparticles through mucosal routes.

Keywords
Nanoparticles · Mucoadhesive · Transmucosal · Toxicity · Fate

Abbreviations HM-EHEC Hydrophobically modified ethyl

hydroxyethyl cellulose
AgNPs Silver nanoparticles HM-HEC Hydrophobically modified hydroxy-
AP-1 Activator protein-1 ethyl cellulose
ARE Antioxidant-response element HPLC  High pressure liquid chromato-
ATP Adenosine triphosphate graphy
BBB Blood brain barrier HSV Herpes simplex virus
BSA Bovine serum albumin IFN Interferon
CBF Ciliary beat frequency IL Interleukins
CeO2 Cerium dioxide JNK c-jun NH2-terminal kinases
CNS Central nervous system KD Kilo Dalton
CNTs Carbon nanotubes LDH Lactate dehydrogenase
CRP C-reactive protein MAPK Mitogen-activated protein kinase
CTAB Cetyltrimethylammonium bromide MgO Magnesium oxide
CuO Copper oxide or cupric oxide MHC Major histocompatibility complex
CVM Cervicovaginal mucus MNPs Magnetic nanoparticles
DCFH-DA 2,7-­dichlorofluorescein diacetate MOCs Mini organ cultures
DISC Death-inducing signalling complex mRNA Messenger ribonucleic acid
DMPC 1,2 Dimyristoyl-sn-glycero-3- MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-­
phosphocholine Diphenyltetrazolium Bromide
DNA Deoxyribonucleic acid MWCNTs Multi walled carbon nanotubes
DPPC Dipalmitoylphosphatidylcholine NF-κB Nuclear factor kappa-light-­chain-
Egg-PC Egg-phosphatidylcholine enhancer of activated B cells
ELISA Enzyme-linked immunosorbent NiO Nickel oxide
assay NPs Nanoparticles
ERK Extracellular signal-related kinases Nrf2  LNT induced NF-E2 p45-related
FAS First apoptosis signal factor 2
Fe2O3 Ferric oxide PbAc Lead acetate
FeO Iron oxide or ferrous oxide PCEP (poly{[(cholesteryl oxocarbonyl-
GFR Epidermal growth factor receptor amido ethyl) methyl bis(ethylene)
GIT Gastrointestinal tract ammonium iodide] ethyl phosphate})
GRO-alpha Growth-regulated oncogene-alpha PCL Poly ε-caprolactone
GSH Glutathione (reduced) PCR Polymerase chain reaction
H2O2 Hydrogen peroxide PEG Polyethylene glycol
Hb Hemoglobin PEO Poly(ethylene oxide)
PLGA Poly(lactic-co-glycolic acid)
3  Transmucosal Nanoparticles: Toxicological Overview 39

PTP Protein tyrosine phosphatases mucin is the main component that is responsible
QD Quantum dot for its viscous and elastic gel-like properties.
qPCR  Quantitative polymerase chain Mucins are large, extracellular glycoproteins with
reaction molecular weights ranging from 0.5 to 20 kDa.
RNA Ribonucleic acid Both membrane bound mucins, and secreted
ROS Reactive oxygen species mucins share many common features. They both
RT-PCR Reverse transcriptase polymerase are highly glycosylated consisting of 80% car-
chain reaction bohydrates primarily N-acetylgalactosamine,
SA Stearic acid N-acetylglucosamine, fucose, galactose, and
SiO2 Silica dioxide sialic acid (N-acetylneuraminic acid) and traces
SOD Super oxide dismutase of mannose and sulphate. Mucin has been difficult
SWCNTs Single walled carbon nanotubes to characterize, due to its large molecular weight,
TAP Diacyl-TAP (l,2-diacyl-3-­trimethy- high polydispersity and high degree of glycosyl-
lammonium propane ation. The conformation of mucin depends on var-
TiO2 Titanium dioxide ious factors such as pH and ionic strength though
TJs Tight junctions sugars also play important role for maintaining
TNF Tumor necrosis factor the extended conformation of mucin [1].
WST-1 Water-soluble tetrazolium salts
ZnO Zinc oxide 3.1.1.1 Mucous and Pharmacology
The physical state of the mucous, change in the
concentration of mucin, and the strong depen-
dence of its physicochemical properties on factors
3.1 Introduction such as ionic strength and pH play a significant
role in many diseases. For example, many bacteria
3.1.1 Overview of Mucosal System possess specific adhesins that specifically bind to
mucous which helps them to reside within the
A mucous membrane is an epithelial tissue layer mucus. This includes pathogenic strains of
lining various cavity of the body and surrounds Helicobacter, Pseudomonas, Streptococcus and
internal organs. It is of ectodermal origin and Pneumococcus. Helicobacter pylori particularly
is incessant with the skin at various body open- resides in the mucus layer of the stomach, and is a
ings such as the eyes, ears, inside the mouth, common cause of ulcers [2, 3]. Some parasitic
inside the nose, the urogenital tract, digestive organisms also secret their own layers of mucus to
tract, and respiratory tract. Mucous membranes escape the immune system. Secondly, overproduc-
are moist due to the presence of glands which tion of mucus is involved in cystic fibrosis, bron-
secrete a thick fluid known as mucous. It serves chitis, asthma and in middle ear infections, and
many functions like lubrication for the passage mucus gels serve as the matrix in which gallstones
of objects, maintenance of a hydrated layer over are nucleated and grow. Whilst, mucus underpro-
the epithelium, a barrier to pathogens and noxious duction is present in dry eye syndromes and in
substances and as a permeable gel layer for the some forms of ulcer disease. Various drug delivery
exchange of gases and nutrients with the under- systems based on mucoadhesive interactions like
lying epithelium. Mucous is primarily composed polyelectrolytic interactions (chitosans, poly-
of water (95%), but also contains salts, lipids acrylic acid, etc.), hydrogen bonds (hydrogels),
such as fatty acids, phospholipids and choles- and disulphide binding (thiomers) have been opti-
terol, proteins which help a defensive purpose mized to increase the residence time. Development
with lysozyme, immunoglobulins, defensins of nanoparticles for mucosal DNA vaccines and
and growth factors. However, the glycoprotein gene therapy are also being underway [4].
40 S. Talkar et al.

3.1.2 B
 rief Discussion on Mucosal fibres arranged in bundles. Due to the composi-
Routes of Exposure tion, physicochemical properties and structure of
the tear film, various factors impact the ocular
Currently many of the treatments depend on sys- mucoadhesion. Very few ophthalmic formulations
temically administered therapies which treat the containing bioadhesives or penetration enhancers
diseased sites but are toxic to healthy tissues lim- are commercially available in the market. The use
iting treatment efficiency due to patient noncom- of bioadhesives considerably extends the corneal
pliance [5]. The advent of micro-and nano-delivery retention time, whereas the absorption promoters
technologies combined with the non-invasive increase the rate and amount of drug transport.
administration brings new confidence for the Combining the two approaches will promise an
treatment of disease. Micro-and nano-delivery increase in the bioavailability [8, 9].
technologies overcomes the problem of drugs and
genes poor solubility, protect drugs from acid 3.1.2.2 Nasal Mucosa
degradation or enzymatic degradation, increase The nasal mucous membrane lines the nasal cavi-
blood circulation, reduce plasma clearance, ties having area of approximately 150 cm2 with
escape the reticuloendothelial system uptake, and highly dense vascular network and relatively per-
achieve higher cellular interaction. Micro- and meable membrane structure, and is adherent to
nano-carriers must increase retention time in the the periosteum or perichondrium of the nasal
mucus to improve the diffusion across the mucus conchae [10]. From the nasal cavity, it is continu-
barrier, which is a challenge in drug delivery. ous with the conjunctiva through the nasolacri-
Positively or negatively charged nanocarriers mal and lacrimal ducts; and with the frontal,
could prolong the retention time in the mucus by ethmoidal, sphenoidal, and maxillary sinuses,
binding forces with negatively or positively through the several openings in the meatuses.
charged mucin glycoproteins. Mucoadhesive drug The mucus layer is 5–20 mm thick and is divided
delivery systems in the past have been formulated into two layers, where the outer layer has a high
as powders, compacts, sprays, semisolids, or films viscosity and a gel-like character, while the layer
[6, 7]. For example, compacts have been used for closest to the cells has a lower viscosity enabling
drug delivery to the oral cavity, and powders and the cilia to move. The turnover time for mucus is
nanoparticles have been used to facilitate drug usually given as 10–15 min, but it is affected by
administration to the nasal mucosa. Recently oral both environmental conditions and diseases [11].
strips were developed for tongue or buccal cavity. Nasal mucoadhesive drug delivery has been
Transmucosal routes covered in the review are under active investigation for controlled release
discussed as follows: dosage forms to deliver drugs directly to the CNS
bypassing the BBB. Drugs administered intrana-
3.1.2.1 Ocular Mucosa sally can travel along the olfactory and trigeminal
Drug administration to the eye is a challenge nerves to reach many regions within the CNS and
because of several clearance mechanisms (tear achieve brain targeting. Although, nasal route of
production, tear flow, and blinking) that protect the administration has gained substantial interest, it
eye from harmful agents. The mucus layer, 40 mm, is limited by the rapid mucociliary clearance,
which is secreted by the goblet cells onto the eye resulting in a limited contact period allowed for
surface, is intimately associated with the glycoca- drug absorption through the nasal mucosa [12].
lyx of the corneal/conjunctival epithelial cells. The
mucus layer is very sensitive to hydration and 3.1.2.3 Oral Mucosa
forms a gel-layer with viscoelastic rheological Drug delivery through the oral mucosa (buccal
properties. It protects the epithelia from damage and sublingual) has gained significant attention
and enables movements of the eyelids. The mucus due to its convenient accessibility. The total sur-
gel entraps bacteria, cell debris, and foreign bod- face area of the oral cavity is approximately
ies, forming “mucous threads” consisting of thick 100 cm2, of which the buccal mucosa represents
3  Transmucosal Nanoparticles: Toxicological Overview 41

approximately one-third. The epithelium of the present in the luminal facet of epithelial cells are
oral mucosa consists of a stratified squamous epi- responsible for the rhythmic upward movement
thelium, the thickness of which varies depending of bronchial secretions within the lung to the
on the site. In the buccal region, the epithelium is pharynx [14, 15].
around 40–50 cells thick, whereas it is somewhat
thinner in the sublingual area. The mucus in the 3.1.2.5 Rectal and Vaginal Mucosa
oral cavity is secreted by salivary glands as a The geometry and morphology of the lower
component of the saliva and is adsorbed to the colorectal canal vary with location, and exhibit
surface of the oral mucosa, forming a 0.1-0.7mm both macroscopic and microscopic features. On the
thick layer. Sublingual mucosa is more perme- macroscopic scale, rectal folds create creases and
able than buccal mucosa, but sublingual adminis- canyons on the mucosal surface. The large folds are
tration is difficult for formulations intended to act found in the relatively short anal canal, which is
over a long period of time [11]. Drug delivery about 2–4 cm long, ending in the anal verge and
through the oral mucosa offers several advan- contains a stratified squamous epithelium. The rec-
tages over other drug delivery systems including tum has crypts on its surface with a thin columnar
bypassing hepatic first-pass metabolism, increas- epithelium having 40–120 μm in diameter and up
ing the bioavailability of drugs, improved patient to about 1 mm in depth. The characteristic length
compliance, excellent accessibility, unidirec- for transport into the mucosal tissue is only about
tional drug flux, and improved permeability [13]. 1 mm, i.e. the thickness of the mucosa.
The oral cavity has been used as a site for local The human vaginal walls are lined with strati-
and systemic drug delivery in different dosage fied squamous epithelium containing numerous
forms like adhesive gels, tablets, films, patches, folds, or rugae, which permit for distension and
ointments, mouth washes, and pastes. increased surface area for absorption. Due to the
intra-abdominal pressure that collapses the rugae,
3.1.2.4 Pulmonary Mucosa high internal surface area, and tortuosity of the
The bronchial wall is made up of mucosa, lamina vaginal canal, to achieve adequate distribution of
propria, smooth muscle, and submucosa with a vaginal product is a challenge. Cervicovaginal
interspersed cartilage. Submucosal glands are mucus (CVM) serves as a physical barrier to pro-
found in the normal human bronchial tree in air- tect the vagina against infection in addition to the
ways with cartilage in the wall. The glands lie epithelium. Mucus produced at the cervix bathes
between the epithelium and plates of cartilage and coats the vaginal walls, mix with vaginal epi-
and between, and occasionally external to, the thelial cells and vaginal transudate. The CVM is
plates of cartilage. The secretory tubules in bron- composed mostly of water (~90 to 95%) with
chial tree arise directly from the collecting duct gel-forming glycoproteins, lipids, soluble pro-
and are usually branched. At the end of each, a teins, enzymes, and various immune factors.
cluster of short tubules is found. Two types of However, ovulatory mucus is produced in more
secretory cells have been recognized in the bron- copious amounts, thus facilitating clearance and
chial submucosal glands lining the tubules, the impeding drug absorption [10, 11].
mucous and serous. Mucous cells line each secre-
tory tubule and its main branches, from the col-
lecting duct to the distal cluster of short tubules. 3.2  ate of Nanoparticles
F
The mucous tubules comprise only columnar via Transmucosal Route
mucous cells with goblet cells and basal cells.
The density of goblet cells progressively Nanoparticles (NPs), when administered via
decreases from the periphery and disappears at transmucosal route aid in efficient delivery of the
the level of terminal bronchioles. The presence of drug without eliciting pain. Drug absorption is
mucin, water, and electrolytes contributes to the higher through a mucosal surface as compared to
solubility of bronchial secretions while the cilia transdermal delivery due to the absence of stra-
42 S. Talkar et al.

Nanoparticles

Protein Corona Retention on mucosal

membranes

Intracellular Transcellular Paracellular

Endocytosis Endocytosis Endocytosis

Degradation/
Drug Release
Clearance

Pinocytosis Phagocytosis

Internalisation

Drug Release

Lysosomal
degradation

Fig. 3.1  Pathways followed by NPs following administration via transmucosal route

tum corneum. Mucosal surfaces are usually rich their contents inside the cell and are then sub-
in blood supply, providing the means for rapid jected to degradation. Figure 3.1 represents the
drug transport to the systemic circulation and pathways followed by Nanoparticles following
avoiding degradation by first-pass hepatic metab- administration via transmucosal route.
olism. However, mucus acts as a barrier to diffu-
sion of lipophilic drugs that interact with the 3.2.1.1 Pathways of Internalisation
glycoproteins and lipids in the mucus [16]. Internalization occurs through intracellular, para-
Nanoparticle (NP) research has proved that NPs cellular, and transcellular pathways [18].
cross mucosal barriers and undergo cellular
uptake. Properties such as size, surface charge, (a) Intracellular endocytosis
shape, hydrophobicity, surface chemistry, and
protein and ligand conjugates affect the phenom- Intracellular endocytosis is of two types i.e.
ena [17]. pinocytosis and phagocytosis.
Pinocytosis is the ingestion of liquid into a
cell by the budding of small vesicles from the cell
3.2.1 M
 olecular and Cellular membrane. Pinocytosis is further divided into
Interactions Clathrin mediated, Caveolae mediated and mac-
ropinocytosis [17]. Clathrin-mediated endocyto-
Following administration, NPs interact with the sis involves clathrin-coated vesicle formation in
mucosal membranes in the vicinity and are either the presence of adaptor and accessory proteins.
internalised into the cell or remain attached to the Signalling of the NP on the cell surface, aligns
mucosal lining and are eliminated without any surface proteins and aids to begin clathrin-­coating
further activity. After internalisation, NPs release on the inner membrane of the cell. An adaptor
3  Transmucosal Nanoparticles: Toxicological Overview 43

protein, Epsin, helps pit formation and accessory 3.2.1.2 Protein Binding to NPs
protein dynamin (GTPase) affects vesicle forma- It has been established through a study by
tion. Thus, a clathrin-coated vesicle with a size of Fleishcher and Payne [21] that extracellular
100–150 nm is formed due to polymerization of serum proteins present in blood get adsorbed
the coat complex. The NP containing clathrin-­ onto the surface of NPs, forming a “protein
coated vesicle then internally detaches from the corona”. When polystyrene NPs functionalized
donor membrane. Once within the cell, clathrin with either amine or carboxylate groups were
and adaptor proteins uncoat to allow fusing of the prepared, serum proteins got adsorbed onto the
vesicle within the cell to release the endocytosed surface of both the NPs. Bovine serum albumin
NPs [19]. (BSA)–NP complexes formed from anionic NPs
In case of Caveole mediated pathway, NPs bonded with albumin receptors on the cell sur-
signalling induces actin reorganization and dyna- face. BSA–NP complexes formed from cationic
min recruitment from the cytosol to stimulate NPs were redirected to scavenger receptors. This
membrane invagination and vesicle budding. The observation that similar NPs with identical pro-
caveolae membrane then fuses into the acceptor tein corona compositions were bound to different
compartment and releases its contents [17]. cellular receptors suggested that a difference in
Macropinocytosis proceeds by forming pro- the structure of the adsorbed protein may be
trusions due to actin polymerization from the cell responsible for the differences in cellular binding
membrane, which then encapsulates the sub- of the protein–NP complexes. Similar results
stance to be internalized and once again fuses were obtained for anionic quantum dots and col-
back with the cell membrane causing internalisa- loidal gold nanospheres. Protein corona remained
tion [17]. bound to the NP throughout the endocytic uptake
Phagocytosis is the ingestion of material by and transport, however, the NP itself altered the
phagocytes and amoeboid protozoans. It involves structure of the adsorbed protein [21].
cell surface recognition followed by sequential
instigation of receptors leading to internalization 3.2.1.3 Degradation
by encircling it into triggered cup-shaped cell Particles generally end intracellularly in endo-
membrane deformations forming a phagosome somes or lysosomes followed by degradation.
[18]. Chemical characteristics such as surface charge
determine the fate of NPs in cells.
(b) Transcellular endocytosis In the case of macromolecular therapeutics,
following intracellular uptake, the contents of the
In this pathway, the NPs to be transported bind endocytic vesicle are delivered to lysosomes for
to the cell membrane receptors and form a com- degradation. Although a therapeutic agent encap-
plex. Membrane invagination is then followed by sulated in NPs are less susceptible to degradation
internalization. The endocytosed vesicle is then in the endo-lysosomal compartment, the rela-
converted into a transcytotic vesicle to prevent tively faster degradation of NPs under acidic
typical endosome degradation. The transcytotic ­conditions in the endo-lysosomal compartment
vesicle is then transported to the other end of the may result in the release of the therapeutic agent,
cell, where the vesicle membrane fuses with the which could then degrade quite rapidly. Thus,
cell membrane and the content of the vesicle is NPs are expected to be efficiently internalized
secreted externally [20]. into the cells and then deliver their payload into
the cytoplasmic compartment rather than be
(c) Paracellular endocytosis retained in the degradative environment of endo-­
lysosomal compartment.
Paracellular delivery of hydrophilic drugs A study by Panyam et al. [22], showed that
occurs through the intercellular space between (PLGA) poly (lactic-co-glycolic acid) NPs were
adjacent cells via tight junctions (TJs). internalized through clathrin depended endocyto-
44 S. Talkar et al.

sis. Following their uptake, NPs were localized in The impetus for designing a nano drug deliv-
the early, recycling endosomes and late endo- ery system is to reduce the toxicity of a drug and
somes and lysosomes. It was summarised that to increase its bioavailability as well as biocom-
NPs are either recycled back to the surface from patibility. On the other hand, their exceptional
the early endosomes or are transported to the sec- properties like surface area to volume ratio, par-
ondary endosomes and lysosomes from which the ticle size, solubility, surface coating and shape or
NPs escape into the cytosol. The early endocytic structure may pose additional risks to the patients
vesicles possess physiological pH wherein NPs [23]. Toxic manifestations observed with in vitro
have a net negative charge and hence are repelled models are hardly relatable to the effects seen
by the negatively charged endosomal membrane. in vivo. Though major entry routes as well as rec-
The secondary endosomes and lysosomes are pre- ognised targets have been identified, intense
dominantly acidic, with pH values ranging from research is still required to demonstrate the path-
4–5. In this pH, NPs have a net cationic potential way and mechanism of toxicity of NPs in the
and hence interact with the negatively charged body [24].
membrane leading to their escape into cytoplas- It is in general consent that NPs display toxic-
mic compartment. NPs do not open up the endo- ity through varied mechanisms and can affect in
lysosomal vesicles but are released by localized allergy, fibrosis, organ failure, neurotoxicity, hep-
destabilization of the endo-­lysosomal membrane atological toxicities, nephrotoxicities, haemato-
at the point of contact with NP, followed by extru- logical toxicities, splenic toxicities, and
sion of the NP through the membrane. PLGA NPs pulmonary toxicities, among others.
are cationic only in the endosomal compartment
and do not destabilize the lysosomes. After their
escape, NPs deliver their payload in the cytoplasm 3.3.1 Physiochemical Properties
at a slow rate, leading to a sustained therapeutic of Nanoparticle and Their
effect. Because NPs are biodegradable and bio- Toxic Effects
compatible and are capable of sustained intracel-
lular delivery of multiple classes of cargoes, they (a) Particle size and surface area: Particle size
are a suitable system for intracytoplasmic delivery and surface area of NPs play an important
of drugs, proteins, or genes [22]. role in their interaction with biological mol-
ecules or system. Interestingly, particle size
is inversely proportional to the surface area
3.3 Mechanism of  Toxicity relative to the volume, means decrease in
at Cellular and Molecular size leads to an increase in surface area to
Level volume ratio. Several biological mechanisms
including phagocytosis, endocytosis and pas-
Nanotoxicity or NP related toxicity implies toxic sive diffusion as well as endocytic process-
effects of NPs which are uncommon and not seen ing (antigen presentation on MHC class
with larger particles on the biological system. molecules) are dependent on size of the
The significance of nanotoxicity is such that even material. One of the prime mechanism for
when the NPs made up of inert materials like toxicity is generation of reactive oxygen spe-
gold or silver, they are highly active owing to cies, these free radicals have been known for
their nanosized dimension. Nanotoxicity pursues hazardous impact on biological molecules
the level or extent to which these properties may like DNA, lipids, proteins etc.
cause any threat to environment and living Furthermore, surface area also results in
beings. It also intends to quantitatively determine some toxic manifestation, i.e. increase in sur-
the severity and regularity of toxic effects of the face area leads to extensive interaction with
exposure of the NPs on the organism. biomolecules that root more oxidation and
3  Transmucosal Nanoparticles: Toxicological Overview 45

DNA damage abilities as compared to larger tive oxygen species than neutral and nega-
particles of same mass [25]. tively charged silica NPs [30].
(b) Particle shape and aspect ratio: Particle (d) Crystalline structure: Besides the three
shape dependent toxicity is related to NPs parameters contributing substantially
made of gold, silver, carbon nanotube, nickel, towards toxicity, crystalline structure may
titanium etc. Endocytosis and phagocytosis also be responsible for nanotoxicity. Studies
processes are mostly influenced by this prop- have claimed that anatase form of TiO2 NPs
erty. It has been shown that spherical parti- induce higher lipid peroxidation and oxida-
cles are more prone to endocytosis than any tive DNA damage in presence of light com-
other particle shapes [25]. Studies also report pared to their rutile form [31].
that particle shape can affect the cellular (e) Aggregation: Particle aggregation also
level e.g. blocking of K+ channel by rod imparts toxicity. Aggregation is mostly
shaped SWNTs were two to three times more dependent on the surface charge, size and
efficient than spherical C60 fullerenes [26]. composition. Aggregation of NPs are mostly
Also in another study nanorod ZnO was seen in the case of CNTs, where it has been
found to me more cytotoxic than spherical observed that aggregated CNTs have more
ZnO [27] cytotoxic effects than dispersed ones [32].
Similarly, greater the aspect ratio more (f) Surface coating: Surface coating eventually
will be the toxicity of NPs. It has been alters the physiochemical properties of NPs
observed that asbestos particles which are such as surface charge, magnetic, electric,
<2  μm in size caused asbestosis, <5 μm optical and chemical properties. These
caused mesothelioma and 10 μm caused lung changes may lead to varied interactions with
carcinoma [28]. TiO2 nanofibres having biomolecules that result into significant toxic-
length of 15 mm were more toxic than fibres ity of NPs. It was well acknowledged that the
having length 5 mm, here former induced existence of ozone, oxygen radicals along
more inflammatory response by alveolar with heavy metals on nanoparticle surface
macrophages in mice than later one. leads to the formation of ROS that induces
Similarly, in case of Carbon Nanotubes inflammation in cells.
(CNTs), long MWCNTs caused inflamma- However, in most cases surface coating could
tory response in mice abdominal cavity also be employed to abate the toxicity of
whereas small MWCNTs did not cause any NP. For example, coating is very essential in
inflammation at all [29]. the case of quantum dots to render them
(c) Surface charge: Surface charge of NPs also ­nontoxic since their metallic core is hydropho-
play an important role in toxicity. Rather, bic and composed of heavy toxic metals like
they have an even greater impact on the bio- cadmium.
logical system. Surface charge on the parti- While these factors mainly contribute to the toxic-
cles dictate various interactions such as ity of NPs, concentration is the principle factor
plasma protein binding, selective absorption, dictating the toxicity of macroparticles [25].
blood brain barrier integrity and membrane
permeability. Mammalian cell membranes
possess negative charge on their surface, 3.3.2 Mechanism of Toxicity of NPs
thereby promoting association of cationic
particles with the cells to a greater extent as Toxic scenario of NPs in organisms and environ-
compared to the negative or neutral particles. ment are well established. Unique physiochemi-
However, higher cationic charge leads to the cal properties of NPs pave way for their
severe toxicity via haemolysis and platelet application in several fields. On the other hand,
aggregation [25]. Positively charged silica they also increase the risk of their exposure to
NPs have been shown to induce more reac- humans and environment.
46 S. Talkar et al.

The mechanisms due to which these proper- which eventually cause the proinflammatory
ties result in toxicity are discussed in the follow- response.
ing section. Eom and Choi [34] reported an elevated ROS
production when Jurkat T cells that were
(a) Reactive Oxygen Species (ROS) or exposed to AgNPs in contrast to the unex-
Oxidative stress production: ROS are oxy- posed ones [34]. Moreover, numerous
gen containing chemically reactive species researchers have established single stranded
that have important roles in cell signalling DNA damage caused due to TiO2, Carbon
and homeostasis. They are generally formed black and diesel exhaust particles [31, 35, 36].
as a natural by-product of oxygen metabo- (b) Apoptosis: It is programmed cell death that
lism. ROS are generated intrinsically as well occurs in multicellular organisms. This pro-
as extrinsically within the cell, the pool of cess involves various events such as chromo-
ROS constitutes of oxidative species includ- some condensation, nuclear fragmentation,
ing superoxide anion (O2−), hydroxyl radical cell shrinkage, DNA and mRNA decay etc.
(OH−), hydrogen peroxide (H2O2), singlet Apoptosis can be initiated by an intrinsic
oxygen (1O2), and hypochlorous acid (HOCl), pathway (cell kills itself because it senses
hypochlorite ion (−OCl−) [33]. cell stress) as well as an extrinsic pathway
NADPH oxidase (NOX) complexes in (cell kills itself because of signals from other
cell membranes, mitochondria, peroxisomes, cells). In case of NPs, they are interacting
and endoplasmic reticulum are responsible with macrophages which causes the activa-
for endogenous production of ROS. Whereas, tion extrinsic pathway of apoptosis. Extrinsic
exogenous ROS can be formed from tobacco, pathway is activated via two signals:
pollutants, smoke, engineered NPs, drugs, (i) Tumour Necrosis Factor (TNF) signal:
xenobiotics, or radiation. TNF-α is a cytokine produced exten-
Enzymes like catalase, glutathione perox- sively by activated macrophages. Human
idase, super oxide dismutase, peroxiredoxins cells have two receptors for it i.e. TNFR1
as well as light are responsible for ROS gen- and TNFR2. Binding of TNF-α to these
eration. Apart from the deleterious effects of receptors leads to the activation of
ROS, they also have some positive effects caspases.
such as programmed cell death i.e. apoptosis (ii) First Apoptosis Signal (FAS): FAS, a
and induction of host defence mechanism. transmembrane protein belonging to the TNF
The harmful effects on biomolecules are family, binds to the FAS ligand. Interaction
often seen in the form of: between them results in the formation of the
(i) DNA or RNA: Single and double death-inducing signaling complex (DISC),
stranded breaks in DNA or RNA which contains, caspase-8 and caspase-10. In
(ii) Proteins: Oxidation of amino acids some cases, caspase-8 is directly activated by
(iii) Lipids: Oxidation of polyunsaturated interacting with foreign materials and subse-
fatty acids i.e. lipid peroxidation quently activate further caspases.
(iv) Enzymes: Inactivation by oxidation of Eom and Choi [34] also reported that 39% of
co-factors Jurkat T cells underwent apoptosis when
NPs may generate ROS via three mechanisms; exposed to the AgNPs [34]. Zno NPs also trig-
particle-cell interaction, active redox cycling gered cell death via Caspase mediated apopto-
on surface of NPs (especially in case of transi- sis as reported by Wilhelmi et al. [37].
tion metals) and oxidative groups functional- Similarly, high concentration of FeO NPs led
ized on NPs [23]. Overproduction of ROS to the 35–40% apoptosis [38].
leads to the activation of interleukins, cyto- (c) Genotoxicity: Nanogenotoxicity is a new
kines, kinase and tumour necrosis factors term that has emerged in the field of nano-
3  Transmucosal Nanoparticles: Toxicological Overview 47

technology. This term highlighted NP Similarly, mitochondrial DNA encodes many

induced genotoxicity and carcinogenesis. structural proteins involved in various path-
Literature showed that, long term inflamma- ways. DNA mutagenesis causes the produc-
tion and oxidative stress present in a cell ulti- tion of defective proteins, thus hampering the
mately leads to DNA damage. Continuous pathways like ATP synthesis, Electron
production of ROS causes mutagenesis (due Transfer Chain, oxidative phosphorylation
to the oxidation, hydrolysis and deamination etc. [33].
of nucleic acid bases, substitution etc.) and Cationic polystyrene NPs induced mitochondrial
carcinogenesis (due to the gene deletion, damage which eventually resulted into cell
insertion etc.) death [40]. Ning et al. [41] asserted that ultra-
The results reported by Eom and Choi fine particles are much potent for induction of
also indicate that DNA damage would have mitochondrial damage [41].
resulted into release of the DNA damage (f) Interaction with cellular signalling path-
marker protein, p-H2AX which increased way: NPs are inclined to induce the signal-
drastically after the exposure of AgNPs [34]. ling cascade pathways via particle induced
(d) Cell surface interaction with proteins: It is release of cytokines, particle to cell interac-
well known that NPs, especially heavy metal tions and binding to the several cellular
NPs tend to interact with proteins and amino receptors as a ligand [33]. Signalling path-
acids. These interactions cause the formation ways which are activated through NPs; for
of protein corona, protein unfolding, and e.g. MAPK, ERK, EGFR etc. are described
altered protein function, which finally culmi- in detail in the following section:
nate into protein damage or non-functional. (i) Nuclear factor kappa-light-chain-­
Similarly, interaction of NPs with protein enhancer of activated B cells (NF-­
molecules such as serum albumin, human κB): NF-κB is a group of proteins that
blood protein haemoglobin (Hb), and cyto- controls transcription of DNA, cytokine
skeletal proteins result in protein conforma- production, cell proliferation, apopto-
tional changes or protein damage. sis, inflammation and cell survival.
Silica NPs significantly influence the These proteins are also responsible for
unfolding of RNase, where the enzyme is the activation of defence mechanisms as
less stable on NPs surface than in free solu- well as cellular responses to stimuli
tion [39]. such as stress, cytokine, heavy metals,
(e) Mitochondrial interaction: Mitochondria is ultraviolet radiation. ROS activate NF-B
the powerhouse of cell, which provides the via the release of IBs resulting in the
energy for vital cell functions. Several toxi- nuclear translocation of NF-B [42].
cological studies shown that NPs have identi- ZnO and CdS NPs were found to
fied mitochondria as a potentially relevant induce toxicity via ROS-dependent
target organelle. NF-B activation [43].
Mitochondrial interaction of NPs lead to (ii) Activator protein-1 (AP-1): Activator
following consequences: protein-1 is a transcription factor that
(i) Disturbance in proton gradient pumps, regulates gene expression in response to
voltage-gated channels a stimuli; including stress, oxidants,
(ii) Change in mitochondrial outer mem- bacterial and viral infection, growth fac-
brane permeability, which releases cyto- tors, and cytokines. AP-1 regulates cell
chrome C (key event in intrinsic pathway proliferation, cell differentiation, and
of apoptosis) apoptosis. This gene is activated via
(iii) Mitochondrial DNA damage, also releases phosphorylation of protooncogene
cytochrome C (results into the necrosis) c-jun. Cr, Ni, and Fe NPs have been
48 S. Talkar et al.

shown to activate AP-1 via ROS genera- pathway such as MAPK, Akt and JNK
tion [44]. leading to DNA synthesis and cell pro-
(iii) Mitogen-activated protein kinase liferation [46].
(MAPK): MAPK is a type of serine/ (vi) Src family kinase: Src family belongs
threonine-specific protein kinase. It is to a non-receptor tyrosine kinases fam-
involved in various cellular responses to ily, which is involved in regulation of
a stimuli; such as proinflammatory cyto- cell growth, cell differentiation and
kines, osmotic stress, heat shock and oncogenic transformation. Oxidative
mitogens. It regulates cell proliferation, stress is responsible for activation of
cell differentiation, apoptosis, gene this family, which later on triggers the
expression, cell survival and mitosis. signal transduction pathway. Src family
MAPK consist of growth factor regu- kinases interact with many membranes,
lated extracellular signal-related kinases cytosolic and nuclear proteins by phos-
(ERK) and the stress-activated MAPK, phorylation of tyrosine [42]. Low dose
p38MAPK and c-jun NH2-­ terminal of Chromium NPs induced cell death
kinases (JNK). MAPK activation is via ROS dependent Src Kinase [47].
based on the oxidative modification of
MAPK signalling proteins (e.g., RTK
and MAP3 K). The concentration and 3.4 Toxicological Aspects
kinetics of ROS production and cellular of Nanoparticles
antioxidant pool are mostly important via Different Transmucosal
for activation of MAPK signalling path- Routes
way. Silver NPs tend to activate JNK
pathway and apoptosis whereas CeO2 3.4.1 Ocular Mucosa-
NPs trigger p38 MAPK signalling in
broncho alveolar cells [45]. In perceiving the advantages of new advances in
(iv) Protein tyrosine phosphatases (PTP): nanobioadhesives for enhancing topical ocular
PTP is a tyrosine kinase that regulates delivery, the other side of the coin must also be
the phosphorylation of various signal- considered. The toxicity literature in this area of
ling molecules involved in signal trans- research is not as robust as other fields, because
duction cascades. Signal transduction most publications focus on the discovery and
cascade pathways are involved in onco- development of new therapeutic agents. It comes as
genic transformation, mitosis, cell no surprise that the same properties that make
growth and cell differentiation. PTP is nanosystems attractive for drug delivery
highly susceptible to oxidative stress in ­applications, may confer reactivity in biological
the form of free radicals and H2O2 [42]. systems and lead to toxicity. For topically ocular
Zn2+ and V4+ NPs are critical in redox administered nanosystems, aggregation and tissue
regulation of PTP via the inhibition of accumulation must be considered. Nanosystem
MAPK and EGFR [46]. aggregation may block cell metabolism and could
(v) Epidermal growth factor receptor impair tissue function. For example, blockage of
(EGFR): It acts as an extracellular pro- the lachrymal drainage punctum and decreased tear
tein ligand for various members of epi- film recycling can occur due to aggregation of topi-
dermal growth factor family (EGF cally applied nanosystems on the ocular surface.
family). EGFR dimerization induces Furthermore, indiscriminate ocular nanosystem
intracellular protein-tyrosine kinase accumulation results in distortion of the ocular tis-
activity which results into auto phos- sue architecture leading to altered function. A very
phorylation. This auto phosphorylation important consideration of toxic effects with nano-
initiates further downstream signalling systems is, that it may be attributable to actual
3  Transmucosal Nanoparticles: Toxicological Overview 49

approaches that are directed to enhance ocular drug pared to the normal dosage forms. Mucoadhesive
bioavailability; the presence of high concentration polymers are being used to increase the residence
of the loaded drug in a non-target tissue. time of formulation within the nasal cavity; the
Chitosan has some unique and important char- polymers possibly interact with the epithelial
acteristics like being mucoadhesive and cationic. tight junctions to facilitate drug absorption by
The positively charged chitosan NPs bind to the some histopathological alterations of the nasal
negatively charged surface of the cornea. Prow mucosa and effect on ciliary beating. Hence it
et al. [48] evaluated Chitosan, PCEP becomes necessary to evaluate the toxicological
(poly{[(cholesteryl oxocarbonylamido ethyl) effects of such nano systems on the structural
methyl bis(ethylene) ammonium iodide] ethyl alterations of the nasal environment.
phosphate}), and magnetic NPs (MNPs) for the Hackenberg et al. [50] evaluated the toxic effect
safe gene delivery in the eye. Rabbits were admin- of repeated exposure of ZnO-NPs in three-­
istered with NPs either intravitreally (IV) or sub- dimensional (3D) mini organ cultures (MOCs) of
retinally (SR) and sacrificed 7 days later. Eyes human nasal mucosa determined by trypan blue
were grossly evaluated for retinal pigment epithe- exclusion and caspase-3 activity, respectively.
lium abnormalities, retinal degeneration, and MOCs were exposed once, twice, or three times to
inflammation. IV chitosan showed inflammation 0.1 or 5 μg/ml of ZnO-NPs for 1 h. per exposure and
in 12/13 eyes, whereas IV PCEP and IV MNPs then evaluated for cytotoxicity and genotoxicity.
were not inflammatory and did not induce retinal DNA fragmentation augmented after 24 h of regen-
pathology. Acute inflammation and polymorpho- eration at both concentrations of ZnO-NPs. In con-
nuclear cell infiltrates resulted with injection of trast, DNA damage induced by the positive control,
these nanoparticle formulations which were methyl methanesulfonate, was significantly reduced
grossly visible in the eye cup after 7 days. after 24-h regeneration. Thus, results suggest that
Histological examination confirmed massive num- repetitive exposure to low concentrations of ZnO-
bers of immune cells at the site of injection. It is NPs results in persistent DNA damage. Various
possible that the hyalocytes, the sentinel immune mechanisms responsible for ZnO-NPs related geno-
cells of the vitreous, are particularly sensitive to toxicity were proposed by the authors based on the
polysaccharides leading to the inflammation [48]. study, such as direct interaction of particles with the
Guo et al. [49] synthesized and evaluated a DNA in the nucleus, ROS generation, or influence
series of positively charged phospholipids and of dissolved zinc ions to DNA damage [50].
cholesterols as membrane components for lipo- Genter et al. [51] assessed the distribution and
somes. Selected liposome preparations formu- toxic potential of 25-nm Silver NPs (AgNPs)
lated with these synthetic lipid materials were (100 or 500 mg/kg) following intranasal (IN)
found to be non-cytotoxic in vitro by using a cell exposure in adult male C57BL/6J mice.
growth inhibition assay, whereas liposomes con- Histopathology of selected organs was per-
taining positively charged components (stearyl- formed, and tissue reduced glutathione (GSH)
amine and cetyltrimethylammonium bromide) levels were measured after 1 or 7 days as an indi-
showed considerable cytotoxicity. Their investi- cator of oxidative stress. Aggregated AgNPs
gations thus, indicate a specific adhesion of the were found in the spleen, lung, kidney, and nasal
cationic liposomes to the surface of mucosal tis- airway by routine light microscopy.
sues owing to the presence of negative charge Autometallography revealed AgNPs distributed
[49]. in olfactory bulb and the lateral brain ventricles.
Elevated tissue GSH levels was observed in nasal
epithelia (both doses at 1 day, 500 mg/kg at
3.4.2 Nasal Mucosa- 7 days) and blood (500 mg/kg at 7 days).
Therefore, intranasal administration of AgNPs
Nasal mucoadhesive drug delivery system offers permits systemic distribution, produces oxidative
the advantage of higher residence time as com- stress in the nose and in blood, and develops
50 S. Talkar et al.

macrophage-mediated erythrocyte destruction in cell viability and permeability at the considered

the spleen [51]. concentrations. At the lowest chitosan concentra-
tion (0.05%) there was no sign of cell toxicity.
However, there was a significant reduction of cell
3.4.3 Oral Buccal Mucosa viability (65%) when the polymer concentration
was increased to 0.1%. At even higher concentra-
The buccal area appears to be an attractive site for
tions (≥0.25%), the cell viability was around 9%;
administration of drugs due to the smooth and representing an almost complete cell death. No
relatively immobile surface, good accessibility, the
significant reductions in the cell viability were
evasion of possible degradation in the gastrointes-observed for the other polymers [53].
tinal tract, and no first-pass metabolism in the Teubl et al. [54] investigated interactions of three
liver. However, continuous salivation and swal- different titanium dioxide (TiO2) particles (i.e. NM
lowing may lead to a very short residence time in 100, NM 101 and NM 105) with oral tissues.
the oral cavity [4]. To overcome this problem, Physicochemical properties were addressed in rele-
novel bioadhesive dosage forms have been devel- vant media, and particle penetration was investi-
oped; such as bioadhesive tablets, patches, bioad- gated with an ex vivo model using porcine mucosa.
hesive gels and ointments, and medicated chewing Although TiO2 particles formed large aggregates
gums. Smistad et al. [52] evaluated the toxicity ofonce dispersed in media, 10–50% remained in the
liposomal formulations on the human buccal cell nanoscale range, rapidly interacting with the mucus
line TR146. The main objective of this paper was layer and infecting the epithelium. NM 100 and
to identify important liposomal formulation fac- NM 105 were found in both the upper part and the
tors influencing the toxicity on cells in the buccal
lower part of the buccal mucosa, while NM 101
region, and identifying significant interactions (smallest particle sizes) only penetrated the upper
between the formulation variables. Positively parts. Transport studies revealed that TiO2 NPs were
charged liposomes were shown to be toxic com- found in vesicles, as well as freely distributed in the
pared to the negatively charged liposomes. Diacyl- cytoplasm. However, NM 105 triggered the produc-
TAP (l,2-diacyl-3-­ trimethylammonium propane) tion of reactive oxygen species [54].
was less toxic than SA, and DPPC was less toxic
than DMPC. The amount of negatively charged
component, the liposome size, and the total lipid 3.4.4 Pulmonary Mucosa-
concentration did not affect the toxicity within the
experimental room. The most toxic combination Pulmonary route serves as a non-invasive
in this study was egg-PC/positively charged lipid approach for systemic delivery of therapeutic
(20 mol%) [52]. actives like drug, proteins and peptides. Larger
Klemetsrud et al. [53] also investigated the surface area of lungs, rich blood supply and a thin
in vitro toxicity, mucoadhesive potential and mucous layer make the pulmonary route popular
impact on cell permeability of polymer coated for efficient systemic delivery. This route is an
liposomes intended for oral use in TR146 cell effective alternative for the delivery of those ther-
line. The following polymers were tested: chito- apeutic actives that show less bioavailability via
san, low-methoxylated pectin (LM-pectin), high-­ oral route, especially in the case of proteins and
methoxylated pectin (HM-pectin), amidated peptides. Specialized devices such as dry powder
pectin (AM-pectin), Eudragit, poly(N-­ inhaler, metered dose inhaler and nebulizer have
isopropylacrylamide-­co-methacrylic acid) been fabricated to facilitate pulmonary delivery
(p(NIPAAM-co-MAA)), hydrophobically modi- by ensuring deep lung deposition. The only sig-
fied hydroxyethyl cellulose (HM-HEC), and nificant formulation based parameter to be con-
hydrophobically modified ethyl hydroxyethyl sidered when designing a dosage form for
cellulose (HM-EHEC). All the systems, except pulmonary delivery is the particle size, the ideal
with chitosan, exhibited no significant effect on size being 1–5 μ. Besides the use of polymeric or
3  Transmucosal Nanoparticles: Toxicological Overview 51

lipidic NPs for relief for pulmonary diseases, field of microbicides and have been advocated
human beings are also inadvertently exposed to for the delivery of promising microbicide drug
inorganic NPs. Metal NPs such as iron, silica, candidates such as dapivirine. Among other
nickel, carbon etc. are inhaled into the body via advantages, nanosystems may be able to enhance
upper respiratory tract in the work place environ- drug/virus interaction, penetrate the mucosa, tar-
ment, which is a common event [55]. In these get HIV-susceptible cells, and provide an effec-
conditions, lung primarily acts as a site of accu- tive and durable drug barrier along the epithelial
mulation and long term exposure of NPs. Once lining when administered by the route. In the
they enter into the interstitial spaces, they are rap- case of vaginal and rectal administration, only a
idly taken up by alveolar cells and induce toxic few studies explored this possibility thus result-
effects. Occupational exposure of these NPs ing in a substantial lack of data supporting the
causes hazardous and harmful effects on human potential value of nanotechnology-based drug
being leading to cancer, asthma, fibrosis, and delivery systems. However early methods for
pneumonitis etc. Naturally occurring NPs are assessing toxic effects of NPs are insufficient to
well tolerated or adapted by human and the envi- detect these toxicities. There is also a paucity of
ronment. However, the unintentional and inten- data regarding excipients, components often
tional inhalation and accumulation of NPs pose a assumed to be non-toxic on the basis of past
serious threat to human as well as environment experience with commercial vaginal products.
[56]. The first report of microbicide toxicity was
Coating of metallic NPs with polysaccharides by Phillips and Zacharopoulos [76] who found
can overcome the drawbacks by increasing sta- that rectal application of N-9 caused rapid
bility and biocompatibility, improving size distri- exfoliation of sheets of epithelial cells and
butions and introducing chemical groups that failed to protect mice against rectal transmis-
allow for further functionalization of the NPs. sion of HSV-2 [76]. das Neves J et al. [77]
Worthington et al. [57] have demonstrated that reported the preparation and characterization
chitosan coating reduced the toxicity of copper of drug-loaded poly ε-caprolactone (PCL),
NPs significantly after 24 and 52 h and the gen- poly(ethylene oxide) (PEO), cetyl trimethyl-
eration of ROS. Conversely, inflammatory ammonium bromide (CTAB) NPs of dapiv-
response of mice exposed to chitosan coated irine, as well as their influence on the
NPs, measured using the number of WBC and permeation and retention in ­cervicovaginal and
cytokines/chemokines in the bronchoalveolar colorectal cell monolayer models, and pig vag-
fluid was shown to increase, as was the concen- inal and rectal mucosa at the cell and tissue
tration of copper ions. These results suggest that level. Dapivirine-loaded NPs of around 175–
coating of metal NPs with mucoadhesive poly- 200 nm and different surface properties were
saccharides (e.g. chitosan) could increase their successfully prepared and were readily taken
potential for use in controlled release of copper up by different anogenital epithelial cells. NPs
ions to cells, but will result in a higher inflamma- were shown able to modulate differently the
tory response if administered via the lung [57]. permeability and monolayer/tissue retention
Tables 3.1 and 3.2 summarises the other in vivo kinetics of dapivirine. PEO-PCL NPs reduced
and in vitro research studies pertaining to the the permeability of dapivirine, while CTAB-
possible toxic effects of NPs via pulmonary PCL NPs increased the diffusion of the drug
route. across studied models. Further, toxicity results
in pig vaginal and rectal mucosa showed unac-
ceptable toxicity with CTAB-PCL NPs as com-
3.4.5 Rectal/Vaginal Mucosa- pared to free dapivirine where it did not show
any toxicity issues [77]. The other toxicologi-
Nanotechnology based drug delivery systems cal cases of the NP drug delivery via vaginal
may be an interesting option to advance in the route are discussed in Table 3.3.
52 S. Talkar et al.

Table 3.1  In vivo NPs toxicity via pulmonary route

No Types of NPs Toxicity Analysis References
1 Diesel exhaust NPs Enhanced gene transcription of interleukin-8 RT-PCR and ELISA [58]
(IL-8) in the bronchial tissue and growth-­
regulated oncogene-alpha (GRO-alpha) protein
expression in the bronchial epithelium
2 Fe2O3 Significant enhancement of free radicals and Histopathology [59]
reduction of the GSH in lung tissue and caused
pulmonary emphysema, interstitial hyperaemia
and inflammation in lungs
3 Multi-wall carbon Induced inflammatory and fibrotic reactions by Histopathology, ELISA and [60]
nanotubes increase production of TNF-α biochemical test
(MWCNT)
4 Silver Dose dependent increase in chronic alveolar Histopathology [61]
inflammation, including thickened alveolar
walls and small granulomatous lesions
5 Silver Disruption in the blood/alveolar epithelial Histopathology and PCR [62]
permeability barrier, oxidative stress and
activation of eosinophils, with release of
cytokines IL-13 and IgE
6 TiO2 dose >5.0 mg/ Developed lung lesions, leakage of cytoplasmic Histopathology [63]
kg contents, compensatory proliferation and
fibrosis of lung tissues
7 Nickel oxide (NiO) Pulmonary inflammation accompanied by Histopathology. western [64]
inflammatory cell infiltration, alveolar blotting and
proteinosis, and cytokine secretion. Increase immunohistochemistry
ROS production induced IL-1β production
8 Cerium Induced a persistent influx of neutrophils and Histopathology and cytokine [65]
Oxide(CeO2) expression of cytokines such as CINC-1, assay
CINC-2, and HO-1
9 Copper oxide Generation of acute neutrophilic inflammation ELISA kit [66]
and cytokine (MCP-1 and IL-6) responses in
the lungs
10 Barium sulphate Dose-dependent lung injury and inflammation Lactate dehydrogenase and [67]
myeloperoxidase assay
11 Silica coated Neutrophilic pulmonary inflammation, elicited RT-PCR and ELISA kit [68]
Titanium dioxide increased expression of proinflammatory
cytokine TNF-α and neutrophil
chemoattractant CXCL1

3.5  ummary and Future

S development of a strategy to evaluate the toxic
Prospects effects of the upcoming nanomaterials and their
products before they are being brought into main-
Nanomaterials are endowed with unique proper- stream use. The toxicity of nanomaterials depends
ties, which are responsible for their toxicological on the basic chemistry of interaction of the mate-
manifestations. This had led to concerns over the rial with the molecular pathways in an organism.
use of such materials, inspite of their widespread A thorough investigation on the molecular interac-
applications. Moreover, rapid developments are tion and alteration in the biochemical machinery
viewed in the field of nanotechnology every day, of an organism upon contact with a nanomaterial
which will only further complicate the toxicologi- is a prerequisite for designing safe and sustainable
cal profile of nanomaterials. This necessitates nanomaterials. There is a multitude of research lit-
3  Transmucosal Nanoparticles: Toxicological Overview 53

Table 3.2  In vitro NP toxicity via pulmonary routes

No Types of NPs Toxicity Cell line Analysis References
1 Diesel exhaust Induces the release of Human bronchial Western blotting [69]
NPs proinflammatory cytokines after epithelial cells
triggering transduction (16HBE)
pathways, NF-κB activation and
MAPK phosphorylation
2 Nanoparticulate Stimulates proliferation which Human bronchial Immunocytochemistry and [70]
carbon black result into autocrine release of epithelial cell line western blotting
EGF and the subsequent EGF-R
transactivation and ERK
cascade activation
3 Silica Dose-dependent increase in A549 human MTT assay [71]
Cytotoxicity and oxidative lung cancer cells
stress
4 Nickel ferrite Dose dependent cytotoxicity, A549 human MTT asaay, qPCR [72]
Induced apoptosis in A549 cells lung cancer cells
through ROS generation and
oxidative stress via p53,
survivin, bax/bcl-2 and caspase
pathways
5 Copper oxide Dose dependent cytotoxicity, Hep-2 Human Alamar blue assay [73]
induce oxidative stress in laryngeal
response to ROS production epithelial cells
6 Copper oxide Dose dependent increase in BEAS-2B-­human MTT assay [65]
cytotoxicity bronchial
epithelial and
RAW 264.7-
murine myeloid
cells
7 Copper oxide Induction of cytotoxic, A549 human MTT assay, neutral red [74]
genotoxic (upregulation of cell lung cancer cells assay, western blotting and
cycle checkpoint protein p53 Catalse, Superoxide
and DNA damage repair dismutase assay
proteins Rad51 and MSH2
expression) and oxidative stress
response
8 Aluminium oxide Dose dependent cytotoxicity A549 and Hep-2 MTT assay [75]
(Al2O3) cell line

erature now available on the toxicity of nanomate- The most common pathways investigated in
rials to various model organisms from human to nanotoxicity experiments are related to oxidative
ecological models. But majority of the reports stress, yet oxidative stress can be a temporary and
focus on acute high dose exposures. Research on natural response to an insult without a negative
the toxicity of other chemicals has shown that dose outcome. Currently, a few individual biomarkers
of a chemical can have a tremendous impact on the are being explored in this capacity and they are
pathways that are affected within the organism. often limited to pathways involved in oxidative
Overall, it appears from the literature that many of stress, which is known to be a complicated bio-
the current categories of available nanomaterials marker. There are a multitude of other potential
are not acutely toxic but are most likely to have non-oxidative stress mechanisms that may be trig-
toxic implications following long-term low dose gered in response to sub-lethal exposures of
exposures. There is a significant gap regarding nanoparticles. Testing these materials at low con-
these types of potential impacts. centrations will allow the development of bio-
54 S. Talkar et al.

Table 3.3  NPs toxicity via vaginal route

No Types of NPs Toxicity Analysis References
1 Silver Silver particles accumulates in vaginal tissue which TEM [78]
caused ultra-structural pathological changes cause
cytotoxic reaction
2 Acyclovir loaded Acute inflammation and high neutrophil infiltration into Standard hematoxylin [79]
polystyrene NPs the lumen and eosin (H&E)
staining
3 Paclitaxel loaded At 24 h after treatment, changes in nuclear morphology Histopathology [80]
NPs (chromatin clumping, nuclear fragmentation) were
evident in a minority of vaginal epithelial cells,
increase in the number of apoptotic epithelial cells was
noted after 48 h
4 Melittin NPs concentrations ≥40 μM mel-NPs resulted in complete MTT assay [81]
loss of vaginal epithelial cell viability

markers for evaluating nanomaterial toxicity. dardized intelligent testing policies should
Also, the ‘unique’ properties demand newer con- become second nature to all ‘nano-scientists/
cepts and methodologies to understand and fore- toxicologists’.
see the size (and shape)-specific interaction of Second, nanomaterials clearly demonstrate
nanoparticles within the human body. Recent the need for alternatives assessment methods to
research has led scientists to identify some dis- consider the intrinsic exposure potential as part
crete physicochemical characteristics of nanopar- of the comparative assessment process because
ticles that required on priority basis to assess their there are distinct physicochemical properties as
toxicological potential. These include particle well as use characteristics that will distinguish
size and surface area in relevant media the route from the bulk material. In this respect the role of
of administration, physical form, degree of aggre- the peer-reviewing process of manuscripts should
gation, surface characteristics, sample purity, etc. be to rigorously demand and enforce these high
Such assessment in the case of nanoparticles standards. All studies that address effects of
poses an additional challenge, since the character- nanomaterials should be reviewed by experts in
istics of nanoparticles largely depend on their the field of nanotoxicology. Moreover, these
form and chemical composition; both exhibiting a experts ought to closely stick to the given
dynamic nature before, during and after adminis- recommendations.
tration into in vitro or in vivo systems.
Also, there is a pressing need for the guidance
and documentation of nanotoxicity which all References
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