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3 In Vitro and In Vivo Tests to Assess Oral Nanocarriers Toxicity
7 Toxicity of Nanodrug Delivery Systems
Table 7.2 In vitro and in vivo parameters to assess oral nanocarriers toxicity
In vitro tests based on cell culture techniques, though with various
restrictions as models for the behavior of cells in an organism, are
very functional in the screening of NPs and in mechanistic assays;
they are comparatively economical and can have a highthroughput
There are enormous assortments of well-known cell lines derived
from different human tissues that keep some of the original
characteristics. A number of them even show the possibility of
differentiation, by means of speciﬁc cell culture conditions, to
better exhibit the characteristics of the organ. Additionally, 3D
cell cultures or co-cultures have also been developed in effort to
mimic the target organ
Various in vitro assays, with diverse toxicological endpoints, have
been projected to evaluate the adverse effect that NPs may
provoke on organs of the human body. Viability assays detect
whether cells are dead or alive, generally by evaluating the cells’
capability to multiply or to form clones. Cytotoxicity assays
examine the consequence of the NPs at different levels within the
cell such as membrane integrity (e.g. lactate dehydrogenase
(LDH) leakage, or oxidative status (e.g. 2070-dichloroﬂuorescein
diacetate (DCFA) assay to detect reactive oxygen species), trypan
blue uptake), metabolic activity (e.g. 3-(4,5-dimethylthia
zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay,
adenosine triphosphate (ATP) detection assay)
Currently, in vitro models, though giving very value data, are
inadequate to predict potential hazards to humans and to
rationalize transition through clinical trials to the market. In vitro
approaches yield incomplete information and they do not
symbolize a realistic model of how NPs will interact with a
speciﬁc organ of the body. For an instance, if the toxic effects of
NPs are connected to inﬂammation, simple in vitro assays may
not be sufﬁcient for examining the toxic potential. Hence
currently information, obtained for in vitro studies involve
veriﬁcation from in vivo experiments to precisely evaluate
Toxicity of Nanocarriers for Oral Delivery
In contrast to the fact that polymeric nanocarriers may offer a number of distinct
advantages over microdevices or macroscopic drug delivery systems, including the
capability to reach some speciﬁc areas or improve the intracellular delivery of macromolecular drugs, they can also produce toxicological issues. In fact, the same
physicochemical parameters determining their fate and efﬁcacy would be involved
in the possibility to induce toxicological effects. However, the main problem is that
most investigations are concentrated on the efﬁcacy of NPs and toxicity aspects, if
studied, are usually restricted to a screening of their cytotoxicity. In this way very
little information is known about the genotoxicity and immunogenic potential of
Toxicity of Nanocarriers for Oral Delivery
Table 7.3 Toxicity considerations of nanocarriers for oral delivery
properties of polymeric
NPs affecting their
Size and shape
Physicochemical parameters of polymeric NPs such as size,
material, shape, surface properties, or the presence of ligands may
result in different “kinetic” properties when administered orally
Various polymers, macromolecules and lipids, both synthetic and
natural, have been employed in formulating biodegradable
nanocarriers. Usually most of these compounds are utilized as
excipients for other pharmaceutical applications or they have the
consideration of “Generally Recognized as Safe” (GRAS) when
administered by the oral route. Nevertheless, as mentioned before,
their conversion into nanoparticulate devices opens the door to
Particle size has obvious effect on the toxicity of nanomaterials. An
inverse relation between size and potential toxic effects is usually
established; small NPs offer a higher surface area and as a result a
higher number of potentially reactive molecules in comparison with
larger ones (given equal mass) . Decreasing the size of NPs
triggers the potential reactivity of these materials in an exponential
For an instance CS-derived NPs as a model of negatively and
positively charged nanocarriers were investigated in their ability to
be taken up by phagocytic cells . Macrophage uptake enhanced
as the surface charge (either positive or negative) increased. This
outcome would be linked to the concern of electrostatic interactions
between particles and phagocytic cells that would allow their
internalization . Nevertheless, when the absolute values of zeta
potential were alike, positively charged NPs offered a higher
phagocytic uptake in contrast to negatively charged ones,
irrespective of their composition 
Theoretically nanocarriers capable of “disappearing” in the
body conditions and/or of being “inert” when in contact with a
living system or tissue would have a lower hazardous effect
[23–26]. Nevertheless, the biodegradability of NPs may produce
biodegradation products with a different toxicological profile
from that of the nanocarrier. In addition, the biodegradation
process, which would occur during the interaction of
nanocarriers with the biological medium, can alter the
physicochemical properties of nanocarriers (e.g. size, shape and
surface properties) and therefore the toxicological performance
may also be influences throughout this process. In contrast,
non-biodegradable materials should present a high toxicity risk
linked with their accumulation in the body
nanocarriers capable of translocating and entering into the circulation after their
administration by the oral route. Similarly, the study of in vivo toxicity has been
pursued only with a small number of polymeric nanocarriers. Various toxicity considerations of nanocarriers for oral delivery is mentioned in Table 7.3.
7 Toxicity of Nanodrug Delivery Systems
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