Review

pubs.acs.org/crt

Biocompatibility of Mesoporous Silica Nanoparticles

Tewodros Asefa*,†,‡ and Zhimin Tao*,§
†
Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, New
Jersey 08854, United States
‡
Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway, New
Jersey 08854, United States
§
Department of Physics, Tsinghua University, Beijing 100084, China

ABSTRACT: In this review, recent reports on the biocompatibility

of mesoporous silica nanoparticles (MSNs) are reviewed, with
special emphasis being paid to the correlations between MSNs’
structural and compositional features and their biological effects on
various cells and tissues. First, the different synthetic routes used to
produce the most common types of MSNs and the various methods
employed to functionalize their surfaces are discussed. This is,
however, done only briefly because of the focus of the review being
the biocompatibility of the materials. Similarly, the biological
applications of MSNs in areas such as drug and gene delivery,
biocatalysis, bioimaging, and biosensing are briefly introduced.
Many examples have also been mentioned about the biological
applications of MSNs while discussing the materials’ biocompati-
bility. The cytotoxicity of different types of MSNs and the effects of their various structural characteristics on their biological
activities, which are the focus of this review, are then described in detail. In addition, synthetic strategies developed to reduce or
eliminate any possible negative biological effects associated with MSNs or to improve their biocompatibility, as necessary, are
illustrated. At the same time, recent reports on the interactions between MSNs and various in vivo or in vitro biological systems,
plus our opinions and remarks on what the future may hold for this field, are included.

■ CONTENTS
1. Introduction 2265
Funding
Notes
2281
2281
Abbreviations 2281
2. Synthesis and Functionalization of MSNs 2267 References 2281
2.1. Synthetic Mechanism of MSNs and their
General Properties 2267
2.2. Functionalization Strategies of MSNs 2267
2.2.1. Stepwise Synthesis (or Post-Synthetic 1. INTRODUCTION
Grafting Method) 2268
Because of their unique physical and chemical properties as well
2.2.2. One-Pot Synthesis (or Co-Condensation
as potential biomedical applications, nanomaterials have long
Synthetic Method) 2268
become the subject of intense research worldwide. Over the
2.2.3. General Properties of Surface-Function-
past two decades, synthetic routes to numerous nanosized
alized MSNs 2268
particles that possess different chemical compositions and
3. Biological Applications of MSNs 2269
physical characteristics and that can induce a diverse range of
4. Biocompatibility of MSNs 2269
biological effects have been developed.1−9 Among many
4.1. Overview 2269
nanosized materials, a class of nanomaterials named meso-
4.2. Effect of Particle Size 2270
porous silica nanoparticles (MSNs) stands out. MSNs have
4.3. Effect of Particle Morphology 2273
tunable nanoscale sizes, different shapes ranging from spheres
4.4. Effect of Mesoporosity and Pore Sizes 2275
to rods, uniform cylindrical mesopores, high surface areas, and
4.5. Effect of Surface Property 2275
easily functionalizable surfaces. Owing to these interesting
4.6. Effect of Cell Type 2278
structural features, the potential applications of MSNs as
4.7. Effect of Methodology of Toxicity Assess-
effective delivery vehicles for pharmaceuticals and bioactive
ment 2278
molecules (e.g., nucleotides) to desired intracellular sites or as
5. Conclusions and Perspectives 2280
Author Information 2281
Corresponding Author 2281 Received: April 16, 2012
Published: July 23, 2012

© 2012 American Chemical Society 2265 dx.doi.org/10.1021/tx300166u | Chem. Res. Toxicol. 2012, 25, 2265−2284
Chemical Research in Toxicology
Figure 1. Proposed LCT synthetic mechanism leading to MCM-41.10,11 The synthetic mechanism can go through two possible pathways as stated in
the text. Reprinted from ref 11. Copyright 1992 American Chemical Society.
host materials for bioimaging, biocatalytic, and biosensing
agents have been widely recognized.5−9
According to the International Union of Pure and Applied
Chemistry (IUPAC), a mesoporous material is defined as a
porous material with pore diameters between 2 and 50 nm. The
first major publications on mesoporous materials were reported
in the early 1990s by two independent groups, namely, Kresge
et al.10,11 and Yanagisawa et al.12 In particular, the reports by
Kresge and co-workers10,11 on the family of highly ordered
mesoporous silica materials called M41S, which consisted of
MCM-41, MCM-48, and MCM-50, had quickly paved the way
to diverse types of ordered MSNs, on whose biocompatibility
this review focuses.
It is important to note here that both the words
nanomaterials and micromaterials have often been used for
MSNs in the literature. This is despite the fact that the
traditional definition of nanomaterials limits the use of the word
nanomaterials only to those materials possessing sizes in the
range of 1−100 nm at least in one dimension. This traditional
definition also emphasizes that nanomaterials should possess
properties that are not extrapolations from a larger size and that
are typically but not exclusively exhibited by materials in small
size ranges. In our opinion, which is possibly shared by many
others, the definition based solely on size is somewhat loose
and not very relevant for MSNs because the properties of
MSNs with sizes <100 nm can be similar to those with sizes
>100 nm. For example, MSNs with sizes of 105 nm or more
often show properties similar to those with 90 nm or less (or as
those defined as nanomaterials in a traditional sense).
Furthermore, most of MSNs have sizes >100 nm in diameter
(typically 60−1000 nm) and exhibit similar structure (e.g., size-,
and shape-)-dependent properties as do other traditionally
defined nanomaterials. So, our use of the word nanoparticles
for MSNs in the review is not to imply that the sizes of MSNs
are strictly <100 nm, but rather to highlight their unique size-
dependent properties (in the same way as do many traditionally
defined nanomaterials).
As research efforts toward the syntheses of various MSNs
and their potential applications in biology and medicine have
intensively continued,13−23 concerns regarding the potential
toxicity of these engineered nanomaterials in human health and
on the environment have also been raised.24−34 Although
several systematic investigations have been conducted to
elucidate and understand the possible adverse effects of
MSNs on biological systems, quite conflicting results have
been documented in recent literature, making the biocompat-
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ibility of these nanomaterials still a subject of intense
debate.35−50
Understanding the compatibility of MSNs within a biological
context requires knowledge of not only the physicochemical
characteristics of the nanomaterials but also the specific
conditions in which the bio-nano interactions take place.51−53
On the one hand, MSNs come with different particle
dimensions, shapes, pore sizes, surface topography, crystallinity,
and mesoporosity as well as functional groups, while at the
same time many more new ones are being reported. In
addition, slight variations in the existing synthetic procedures
and postsynthetic modifications of MSNs can result in an array
of MSNs with different structural and surface properties. On
the other hand, the complexity of biological conditions under
which the nanoparticles are applied can also lead to a range of
unpredictable biological responses to MSNs.
Many of the biocompatibility issues associated with MSNs,
or nanomaterials in general, are mainly to do with whether and
how the nanoscale materials interfere with a variety of
biological processes. At a cellular level, the potential
cytotoxicities of MSNs could result from intracellular injuries
caused by the interaction of MSNs with the biological systems
through various complex mechanisms, including membrane
peroxidation, glutathione depletion, mitochondrial dysfunction,
and/or DNA damage. How specifically these processes are
impacted by MSNs or how MSNs interact with biological
systems varies depending on the type of cell or MSN in
question. Moreover, the possible toxicity of a given MSN after
the nanoparticle enters the animal’s or the human body could
manifest itself in the form of impairments in lung, brain, skin,
blood circulation, immune system, etc. Thus, putting together
all of these variables involving the materials as well as the
biological systems, a complex set of biocompatibility scenarios
for these nanosized particles (i.e., MSNs) can be expected.
Nevertheless, despite these complexities, because of the
promising applications of MSNs in biological and medical
fields, it is still vital to obtain sufficient information about their
possible biocompatibility and the factors that affect their
biocompatibility in order for these materials to be further
advanced for clinical use.
In this review, we first introduce the synthetic routes used to
produce MSNs and the surface functionalization methods
employed to modify their surfaces and compositions. We then
briefly illustrate the applications of MSNs for drug and
biomolecular delivery, bioimaging, and biosensing. Next, by
categorizing the most important physiochemical properties of
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Table 1. Advantages and Disadvantages of the Two Commonly Adopted but Different Synthetic Methods to Functionalize
MSNs
method advantages
postgrafting (step- Mesostructures and mesoporosity of parent MSNs basically
wise) synthesis remained.
grafting of the organic groups effectively performed on the available inhomogeneous distribution of the functional groups with usually higher
surfaces on the MSNs
co-condensation a direct synthesis of MSNs with functional groups at the same time simultaneous addition of functional molecules co-condensed with the
(one-pot) as the orderly mesostructures with certain morphology formed
synthesis
the synthesized periodic mesoporous organosilicas was chemically
diversified by the insertion of nonhydrolyzable Si−C covalent
bonds
MSNs, we systematically discuss the effect of the most
important physiochemical features of MSNs on biological
processes, while at the same time we describe many of their
potential biological applications using different examples. We
then discuss the various physical and chemical methods
employed to minimize the possible cytotoxicity of these
nanomaterials. Finally, we elaborate the different types of
biological assays used to measure the biological activities of
nanomaterials, including MSNs, and their implications.
2. SYNTHESIS AND FUNCTIONALIZATION OF MSNS
2.1. Synthetic Mechanism of MSNs and their General
Properties. The synthesis of MSNs is achieved via a process
called supramolecular self-assembly (also called soft-templat-
ing), which involves two major steps: (1) the hydrolysis and
condensation of different silica precursors such as tetraethox-
ysilane in the presence of ordered assemblies of surfactant
micelle templates and (2) the removal of the surfactant
templates by calcination or solvent extraction to generate the
ordered mesoporous silica structures. The resulting mesopo-
rous silicas or MSNs have regular arrays of uniformly organized
mesochannel pores, whose pore sizes can be tuned simply by
using differently sized surfactants as templates.
Two mechanisms have been generally proposed to describe
the synthesis of MSNs. The first one involves liquid crystal
templating (LCT) (Figure 1) that proceeds through the
following five steps: (1) formation of surfactant micelles in
solutions; (2) organization of the surfactant micelles into
cylindrical micelles; (3) stacking of the cylindrical micelles into
a regular array of micelle liquid crystals; (4) adsorption of
anionic silicates onto the positively charged surfaces of the
micelle liquid crystals; and (5) removal of the surfactant micelle
templates to produce the mesoporous silica structures.10−12
The second and alternative pathway involves a cooperative
assembly, which combines steps 1−3 above into a single
concerted process that leads to a regular array of surfactant−
silica assemblies. This is followed by steps 4 and 5 mentioned
above. According to this model, the resulting liquid crystal
structures can be reshaped by changing the reaction conditions
during the synthesis of the materials, such as by varying the
value of ionic strength, the type of silicate precursors or
cosurfactants, the number of ionic charges on the silicate
precursors, the concentration of surfactants, etc.10−12 In
addition to these two general mechanisms, other mechanistic
studies and information to explain the formation of MSNs have
been proposed by many other research groups.54−56
Regardless of the exact mechanism involved during their
synthesis, the resulting MSNs are typically spherical, rod, or
oval shaped and have mesoporous structures; uniform nano-
meter pores (typically between 2 and 15 nm); large surface
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disadvantages
nonselective deposition of functional groups on the inner and outer
surfaces of the MSNs
density at the pore mouths and on external surface of the MSNs
mesophase of template−silicate aggregates, affecting the structural
features of resultant MSNs
only organic extraction adopted as a postsynthetic treatment of
functionalized MSNs to remove the templating surfactant
areas (typically ∼1000 m2/g or even more); and large pore
volume (up to 1 cm3/g).17−22 Furthermore, their pore
structures can be in well-ordered hexagonal or cubic arrange-
ments and thus can give diffraction patterns on powder X-ray
diffaraction, even though their silica frameworks are almost
always amorphous. By changing the reaction conditions, MSNs
can be synthesized with various morphologies and different
sizes ranging from tens to hundreds of nanometers. While
MSNs are generally hydrophilic, thanks to their residual surface
silanol groups, they can also easily become hydrophobic by
functionalizing their surfaces with various organic groups, as
discussed in the following section. All these structural and
compositional variables can thus make MSNs (1) highly
versatile for various applications; (2) have diverse ranges of
biological activities (ranging from highly biocompatible to
highly toxic) toward different cell lines and tissues, in vivo or in
vitro; and (3) have tunable biological responses (e.g., MSNs can
be made highly biocompatible by modifying their surfaces with
rationally chosen functional groups).
2.2. Functionalization Strategies of MSNs. As men-
tioned above, MSNs can be synthesized with different structural
features, including particle sizes, shapes, surface areas, nanoscale
pore sizes, pore volumes, and surface-modifying groups on their
internal or external surfaces, or even both. It was also indicated
that by altering their structures or surface functional groups, the
physical and chemical properties of MSNs can easily be tuned.
The ease of tuning their surface properties can further be
exploited to make MSNs biocompatible, carry payloads of
hydrophilic or hydrophobic drugs, or target specific cells or
intracellular sites. Surface-functionalized MSNs are widely used
in biological applications because many such materials,
especially those containing judiciously chosen functional
groups, have improved adsorption capacities to bioactive
molecules or drugs, increased binding abilities to specific cell
and tissue targets, enhanced overall biocompatibility, etc.
Hence, we here introduce the most commonly used synthetic
methods employed for surface functionalization of MSNs and
discuss the advantages and disadvantages of each synthetic
method (see Table 1).
The surface functionalization of MSNs is typically achieved
by introducing functional groups either on the external or
internal surfaces of the MSNs or, in most cases, on both
surfaces. This can be carried out through one of the two basic
synthetic strategies: stepwise synthesis (postsynthetic grafting)
or one-pot synthesis (co-condensation method). By using
either of the two methods, monofunctional or multifunctional
MSNs can be produced, depending on the types of functional
groups (one or more than one type, respectively) introduced
into the MSNs. Furthermore, the functional group(s) can or
should be rationally chosen in order to form functionalized
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Chemical Research in Toxicology
MSNs with desired surface properties, morphology, pore size,
and structures and thus suitable biocompatibility for medical
applications.
2.2.1. Stepwise Synthesis (or Post-Synthetic Grafting
Method). The stepwise synthesis or postsynthetic grafting
method to functionalized MSNs generally comprises two-steps:
(1) synthesis of as-made MSNs and (2) postsynthetic
modification of their surfaces with functional molecules,
typically organosilanes. This synthetic strategy was actually
first adopted immediately after the first reports on the syntheses
of ordered MSNs in the early 1990s.
The postsynthetic grafting method has some advantages as
well as disadvantages compared to the one-pot synthetic route
(see Table 1). The advantages of the postsynthetic grafting
method include (1) the MSNs retaining their mesostructures
and mesoporosity to a large extent after the introduction of the
functional groups and 2) the grafting of the organic groups that
can easily be performed on the available surfaces of the MSNs
with this synthetic method.
The disadvantages of the one-pot synthetic route include (1)
the synthesis results in the nonselective deposition of functional
groups on the inner channel walls as well as the outer surfaces
of the MSNs and (2) the final materials containing
inhomogeneous distribution of the functional groups, with
higher density around the pore mouths and the external
surfaces of the MSNs.
It is worth noting that the effectiveness of the postgrafting
functionalization could be largely dependent on how the
surfactant templates from the as-prepared mesostructured silica
are removed or how the parent MSNs are prepared. Generally,
two methods are employed to remove the surfactant templates
from the as-made mesostructured silica materials, i.e.,
calcination and solvent extraction. If calcination is used to
remove the surfactant templates, the density of surface silanol
groups in the resulting MSNs becomes lower because the
higher calcination temperature favors silanol condensation in
the materials.57 The lower silanol density, in turn, leads to less
reactivity of the MSNs with organosilane surface modifiers and
ultimately lower density of surface organic groups in the
functionalized MSNs.57
In the case of solvent extraction, the as-prepared
mesostructured silica nanoparticles are stirred in acidic
solutions in order to replace the surfactant templates with
protons. Besides producing a porous structure, this process
leaves more silanol groups on the pore walls of the resulting
MSNs. Nevertheless, calcination is still a preferred route to
prepare MSNs for biological applications, as it forms MSNs free
of any residual organic templates that are often detrimental to
biological systems.
In addition to the specific method used to remove the
surfactant templates, the reaction conditions such as pH,
temperature, and solvents used during the synthesis of the as-
made mesostructured silica materials can affect the surface
coverage of functional groups during postgrafting of the MSNs
with organosilanes.43,58,59 Not surprisingly, the physical proper-
ties such as the morphology, pore size, and surface area of the
as-made MSNs (the parent materials) can also influence the
surface distribution and the density of organic surface modifiers
on the functionalized MSNs.
2.2.2. One-Pot Synthesis (or Co-Condensation Synthetic
Method). In the case of the one-pot synthetic method, also
known as co-condensation synthesis, organosilane surface
modifiers, the surfactant templates, and the silica sources are
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mixed together to make the as-prepared organic-functionalized
mesostructured silica nanoparticles in one step. As the self-
assembly and co-condensation processes take place and the
mesostructured material forms, the functional groups from the
organosilanes are incorporated directly into the material.
However, the addition of the functionalized silanes into the
typical silicate/template solution could affect the self-assembly
process as well as the structural features of the final materials,
including the mesoporosity and degree of ordered structure of
the MSNs. Thus, care should be taken while choosing the
concentrations of the organosilanes during the synthesis of
organic-functionalized MSNs with this method. Upon extrac-
tion of the surfactant templates from the resulting materials,
this synthesis leads to the formation of MSNs with functional
groups exposed on their outer surfaces.
Compared with the stepwise postgrafting synthetic method,
the co-condensation synthetic method offers a more convenient
approach to surface modify MSNs with organic functional
groups in one pot, while producing a rather homogeneous
distribution of functional groups. However, in the case of the
organic-functionalized MSNs synthesized with co-condensation
method, the surfactant templates can be removed only by
solvent extraction (or they cannot be removed by calcination)
in order to preserve the organic functional groups introduced
into the mesostructured materials.
2.2.3. General Properties of Surface-Functionalized MSNs.
MSNs are generally modified by attaching suitable functional
groups on their surfaces via the one- or multistep synthetic
methods mentioned above. By using either method or their
combinations, the internal and/or external surfaces of the
surfactant-extracted MSNs can be functionalized with the right
organic groups, and the surface charges or hydrophilicity/
hydrophobicity of MSNs can be tailored. Given the value of the
isoelectronic point of silica (including MSNs) of ∼2.0 and their
acidity in physiological pH of ∼7.4 or under most pathological
conditions (e.g., pH ≤ 7.0 in malignant tumors), pure MSNs
remain negatively charged in most biological environments.
Thus, tuning the exterior surface charge and chemical affinity
judiciously by surface modification with organic groups is very
useful and sometimes necessary in order for MSNs to serve as
effective host materials for drug or bioactive molecules,
biosensors, biocatalysts, or site-specific bioimaging agents.
As briefly mentioned above, the surface properties of
functionalized-MSNs can be different depending on how the
surfactant templates of the parent MSN material are removed,
i.e., whether solvent extraction or calcination is used to remove
the surfactant templates. Both types of template extraction
methods have their own advantages and disadvantages. Solvent
extraction, for example, produces MSNs under mild conditions
without degrading the organic templating agents to be further
recycled, if necessary. However, solvent extraction also
produces MSNs that may still have some residual templating
agents inside their mesopores. These residual surfactant species,
which are often cationic and which could remain on the MSNs’
framework through electrostatic or hydrogen bonding inter-
actions, can be detrimental to the integrity of cell membranes
or intracellular organelles due to their lipophilic nature. In
addition, the repeated exposure of the MSNs to organic
solvents during solvent extraction may result in possible
accumulation of free radicals in the solvent-extracted MSNs
and unexpected negative consequences toward cells.44
However, calcination results in MSNs that are free of
possible residual surfactants and that are generally preferable to
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Figure 2. Transmission electron microscope (TEM) images for MCM-41 and SBA-15 MSNs.40 Reprinted from ref 40. Copyright 2008 American
Chemical Society.
Figure 3. Various potential applications of MSNs in biological systems.
their solvent-extracted counterparts for biological/medical
applications. However, the removal of the organic groups by
calcination also produces MSNs with pristine silica or
negatively charged surfaces, which could induce electrostatic
repulsions of the MSNs by the negatively charged cell
membranes and inhibit the endocytosis of the MSNs.44,60
While the calcined MSNs may benefit from their increased
siloxane cross-linkages as a result of the heat treatment, these
materials sometimes suffer from the partial collapse of local
mesostructures. The latter, in turn, may lead to possible
degradation of the MSNs into some polysilicic acids, which can
denature bioactive molecules (e.g., proteins) and induce severe
cytotoxicity.44
3. BIOLOGICAL APPLICATIONS OF MSNS
Owing to their mesopore sizes, large pore volumes, and high
surface areas, MSNs (Figure 2) can serve as suitable host
materials for many types of pharmaceuticals (e.g., anticancer
drugs) and biologically active molecules (e.g., proteins and
nucleotides). Moreover, their highly ordered mesoporous
structures and large surface areas as well as their ease of
surface functionalization make MSNs ideal for adsorbing and
holding up large payloads of drugs or bioactive substances
inside their mesopores. In addition, the dimensions of MSNs
can be tuned in the range of 60−1000 nm, which is a suitable
size range for passive delivery of drugs into cells. Furthermore,
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many MSNs at certain dosages and with specific morphology
and functional groups are proven to be biologically
friendly.5,6,8,9 Therefore, it is not surprising to see that MSNs
have been intensively investigated for different biological or
medical applications. In fact, several papers with many
interesting results, including comprehensive reviews, on the
potential applications of MSNs in different biological systems
for drug delivery, bioimaging, biosensing, biocatalysis, and more
recently, for theranostics (Figure 3) have been well
documented.5−9
4. BIOCOMPATIBILITY OF MSNS
4.1. Overview. As described above, MSNs have interesting
structural features that can be exploited for different biological
applications. These structural features include their nanoscale
sizes, mesoporous structures, high surface areas, large pore
volumes, and easily tunable surfaces. Furthermore, owing to the
nature of their composition, i.e., silica, MSNs are more stable
systems under various biological environments compared with
many other biomaterials such as polymers. In addition, many
types of MSNs have been shown to be nontoxic in many
biological systems if they are prepared with certain optimized
structural features and are applied at the right dosages.5,6,8,9 For
these reasons, MSNs have been pursued vigorously in the past
two decades and have long emerged as candidate materials for
an array of biomedical applications. However, despite many in
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Figure 4. (a) Cellular uptake of differently sized MSNs.62 HeLa cells were treated with 100 μg/mL of 30, 50, 110, or 170 nm in diameter MSNs
containing green fluorescent tags. Cell skeleton and nucleus were stained in red and blue, respectively, as shown. The amount of silicon per cell was
calculated and plotted as a function of size of MSNs. Reprinted with permission from ref 62. Copyright 2009 John Wiley and Sons. (b) Energy offset
during the interaction between MSN and cell membrane,65 demonstrating the size- and surface-dependent interaction of MSN (presumed to be
spherical shaped with the radius = r) and RBC membrane. Reprinted from ref 65. Copyright 2011 American Chemical Society.

vivo or in vitro studies, including some using animal models and
tissues, clinical tests with MSNs in humans have not yet been
conducted.42 This is because our understandings of the full
ranges of biocompatibility of MSNs in various biological
systems still remain far from complete. This, in turn, is due to
the fact that the biological effects of such nanomaterials are
complex as they generally rely on a range of nanoscale features
of the materials, such as their particle sizes, shapes, dosages,
pore structures, composition, etc. as well as the type of
synthetic method used to make them. Given these different
physicochemical characteristics of MSNs, the overall interaction
and eventual fate of these engineered nanomaterials within
biological systems are difficult to fully probe. Thus, further
efforts on detailed and systematic studies to determine what
specific structural factor(s) of these nanomaterials is (are)
responsible for their biological effects under various conditions
need to be continued. Only then can these potentially useful
nanomaterials for biology and medicine possibly enter human
clinical trials in the future.
As discussed in the Introduction section, various synthetic
strategies have been developed to produce MSNs with different
sizes, shapes, pore structures, etc. This has actually been among
the major strides in the field of material chemistry over the last
two decades. However, this development has also led to the
growing questions regarding the potential biological effects of
MSNs. Specifically, the success in nanomaterial synthesis has
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invigorated researchers’ interests to investigate how the size,
shapes, and many other structural features of nanomaterials in
general and MSNs in particular impact their biological activities,
such as their endocytosis through cell membranes, interference
within cellular signaling, interaction with cellular organelles, etc.
4.2. Effect of Particle Size. The size of MSNs is often
implicated as being one of the most important factors that
determine whether MSNs result in the desired or unwanted
effects on biological systems, i.e., whether the MSNs become
biocompatible or severely toxic upon physical contact with
biological entities. The size of particles is also an important
determinant factor for the biocompatibility of many other
nanomaterials. Generally, MSNs cannot be regarded as simple
matrices for auxiliary biomedical uses since many nano-
structured materials, including some MSNs, have long been
known to inherently interfere with cellular processes.60−63 In
addition, the size as well as other structural features of
nanomaterials has been shown to influence how the nano-
materals interact with cells or some cellular processes. The
relationship between the sizes of a given nanomaterial, such as
MSN, and their biological activity cannot, however, be fully
discussed without bringing at least the dosage of the
nanoparticles into the picture. In other words, the maximum
amount of nanoparticles that can cause no noticeable biological
response (or the minimum dosage that can lead to cell death)
can vary for a given nanoparticle depending on its size.
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Chemical Research in Toxicology
Therefore, as the effects of a given physiochemical feature of
MSNs (e.g., size) on their biological responses are discussed,
(1) at least the dosages of the MSNs applied in each specific
case may have to be included in the discussion, and (2) at most,
one or more of the other structural features of the MSNs such
as the shape, pore size, or pore volume may have to also be
included in the picture.
The effects of size of MSNs on cellular uptakes and function
were studied in detail by Vallhov et al. using different types of
MSNs.61 Specifically, the authors synthesized two anionic
surfactant-templated mesoporous silicas (AMS), i.e., AMS-6
(3D cubic cylindrical type MSNs with particle size = 270 ± 50
nm) and AMS-8 (3D cubic cage type MSNs with particle size =
2500 ± 500 nm), by using calcination to remove their
templates. The authors then investigated the biological effects
of the MSNs by incubating the nanoparticles with human
dendritic cells (DCs). When comparing MSNs with similar
surface areas in the range of 520−550 m2/g, those with smaller
sizes or at lower dosages affected the immune functions, cell
viabilities, and particle uptakes of DCs less than those with
bigger sizes or at higher dosages.61 Furthermore, although these
differently sized particles entered the DCs, presumably through
similar mechanisms, the larger ones were found to escape from
the endolysosome more easily than the smaller ones.61 In
another study, Mou and co-workers synthesized ordered
monodispersed MSNs with uniform sizes in the range of 30
to 280 nm (most of which had hexagonally ordered structures,
except for the 30-nm sized MSNs, which had predominantly
worm-like mesostructures), and they then investigated their
internalization by HeLa cells.62 While the cell proliferation and
viability were found to be unaffected at a dosage of 100 μg/mL
for all the MSN particles despite their differences in size, their
cellular uptakes varied with sizes in the order of 50 > 30 > 110
> 170 nm (Figure 4a).62 These results clearly indicate that
endocytosis of MSNs is virtually a complicated process,
determined by many more factors than just the particle size
of the MSNs. In a recent report, the impact of particle sizes of
MSNs on cell adhesion and migration was investigate by
incubating 100 μg/mL of spherical MSNs with 80 and 500 nm
in diameter with human dermal fibroblast cells.63 Owing to
their similar surface charges as seen from their ξ-potential
values, both types of MSNs were found to be internalized by
the cells via similar mechanisms, which involved macro-
pinocytosis, the clathrin-mediated pathway, and to a lesser
extent, the caveolae-mediated process; however, the larger
particles were found to be ingested more quickly and
accumulated more in lysosomes than their smaller counter-
parts.63 Furthermore, when the dosage of the MSNs was
increased from 5 to 200 μg/mL, the 80-nm size spherical MSNs
inhibited cell growth and proliferation more than did the 500-
nm MSNs, although the dose-dependent effect was exhibited by
both types of MSNs. These biological effects were results of the
severe collapse of the mitochondrial membrane potential,
decrease in dehydrogenase activity, and damage to the
membrane integrity. Conversely, the 500-nm size MSNs
reduced mRNA levels of major adhesion proteins (i.e.,
fibronectin, laminin, and focal adhesion kinase) less than did
their 80-nm size counterparts.63 However, both types of
particles seriously weakened cell migrations, possibly due to the
presence of strong interactions between these nanoscaled
particles and nucleotides.63
To evaluate the effect of size of MSNs on hemolysis, the
Haynes group synthesized different batches of MCM-41 type
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MSNs with sizes ranging from 25 to 225 nm, while keeping
their structures the same (i.e., hexagonal or worm-like) and
their surface areas similar (i.e., BET surface area = ∼1038−
1164 m2/g).64 When examining the particles’ interactions with
human red blood cells (RBCs), they found that these MCM-41
type MSNs produced size- and dose-dependent hemolytic
effect, with the smallest size MCM-41 MSNs or the largest
dosage exhibiting the highest toxicity.64 The authors also
showed that the size-dependent hemolysis was present only
when the nanoparticles had long-range ordered porous
structure.
In another related study, Zhao et al. recently showed that the
bigger size SBA-15 type MSNs (∼531 nm) were engulfed more
by RBCs and caused greater membrane distortion in the cells
than their smaller size counterparts, MCM-41 type (∼122 nm)
MSNs.65 The adsorption of the larger SBA-15-type MSNs to
RBCs induced a stronger local membrane deformation leading
to more internalization of the particles and ultimately more
hemolysis. On the contrary, the smaller MCM-41-type MSNs
were adsorbed onto the surface of RBCs without disturbing the
membrane or morphology. To explain these results, Zhao et
al.65 proposed that the engulfment of nanoparticles by RBCs is
determined by the combined effect of two competing
processes: (1) an exothermic process involving covalent bond
formation between the surface silanol groups of MSNs and the
phosphatidylcholine groups of RBC membranes; this inter-
action releases more binding energy when the cells capture the
bigger size MSNs with larger external surface areas than the
smaller ones which have less external surface areas; and (2) an
endothermic process resulting from the entrapment of MSNs
by RBC membranes; this process requires a larger degree of
bending energy for the smaller particles than the bigger ones
because the relatively more flat cell membrane should undergo
more changes in its orientation to accommodate the smaller
particles than the larger ones. The combined effects of these
two competing processes could also explain well why the
ingestion by RBCs of the larger size SBA-15 MSNs having
higher external surface area of 155.4 m2/g was thermodynami-
cally more favorable than the ingestion by RBCs of the smaller
size MCM-41 MSNs possessing lower external surface area of
81.6 m2/g.65
It is worth noting here that energy offset during the
interaction between MSNs and cell membranes could be used
to explain the effect of the size of MSNs on the degree of their
cellular internalization, in which many energy-dependent routes
might simultaneously be at play (Figure 4b). Furthermore, each
uptake pathway for MSNs could be modeled and fit into a
single function by taking into consideration many of the
variables associated with the cells as well as the MSNs including
particle size, particle shape, surface chemistry, cell type, etc. The
final gain or loss in energy as a result of the endocytotic process
under each specific circumstance then dictates whether the
contact/interaction between the cells and the particles would
be favorable or not. This concept could also help clarify many
of the previously reported contradictory results regarding the
underlying mechanisms of cellular internalization of nano-
particles and in some cases their subsequent biological effects as
well. For example, for MSNs with different sizes but similar
BET surface areas, the smaller ones, which usually have
relatively higher external surface area per unit mass, may cost
the cells more energy when the cells engulf these particles,
which may lead to more cell injury and death.64
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Figure 5. (a) Biodistribution of MSNs in a mouse model with xenograft tumor:46 (i) the percentage of silicon in urine and feces of mice several
hours after injection of the MSNs through the tail vein of mouse compared to the total amount of material injected into the mouse; (ii) nanogram of
silicon found per milligram of murine tissue collected several different times after injection of the MSNs.46 Reprinted with permission from ref 46.
Copyright 2010 John Wiley and Sons. (b) Antitumor effects of MSNs loaded with CPT. Reprinted with permission from ref 46. Copyright 2010
John Wiley and Sons. (c) Scheme showing accumulation of MSNs in cancer tissues by to EPR effect.76 Reprinted with permission from ref 76.
Copyright 2009 John Wiley and Sons.

In addition to RBCs, MSNs' interaction with white blood
cells (WBCs) has also been studied. As part of the body’s
immune defense system, WBCs (sometimes called phagocytes)
are known to engulf foreign particles > ∼100 nm in diameter
upon encountering them.64 In some cases, active phagocytes
would agglomerate into giant phagocytic cells in order to engulf
large or (sub)micrometer sized foreign particles. Being part of
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the reticuloendothelial system (RES), mononulcear phagocytes
are originally derived from bone marrow and widely migrated
into a variety of organs through blood circulation, including
brain, lung, heart, lymph nodes, liver, spleen, and subcutaneous
tissues. Depending on their dimensions, particles, whether
intercepted by RES or not, flow through the bloodstream and
can reach the aforementioned tissues. While some of the
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Chemical Research in Toxicology
particles accumulate in some of these tissues, others get
excreted either via renal clearance with urinal excretion or
hepatic bile with feces. The residual free particles remain in
circulation in the bloodstream or get trapped by vascular walls
or other substances in the fluid (e.g., platelets, proteins).65 On
the basis of these results, we can infer here that the size of
nanoparticles can affect the distribution of those particles in
vivo.
Given the potential biomedical applications of MSNs such as
their use as drug delivery agents, the compatibility of MSNs to
blood deserves special attention. It was recently reported that
nanomaterials could rapidly interact with platelets and
endothelial cells residing along the inner surface of blood
vessels and cause platelet aggregation and thrombosis.66
However, no study has shown that MSNs exhibited any toxic
effects on platelets, while MSNs were used to control
hemorrhages in the same way as other materials such as
zeolite-based composite materials were able to do so.67,68 On
the other hand, other silica-based69−71 or porous nanomaterials
(e.g., carbon nanotubes)66,72,73 were shown to exhibit
immediate effect on inducing platelet aggregation.
Recently, the Radomski group examined the impact of dense
and amorphous silica nanospheres (10−500 nm in diameter)
on the human vascular endothelium and platelets.69−71 The
silica nanoparticles with smaller size induced more oxidative
stress on primary human umbilical vein endothelial cells
(HUVECs) in a concentration-dependent manner, converting
NO into ONOO−, which activated NF-κB and then initiated a
cascade of immune responses that finally led to cell death.69 A
reduced [NO−]/[ONOO−] ratio was also found in platelets
that were treated with these nanosize silica particles.71 This
biological effect by these silica nanoparticles was probably the
result of nanoparticle-induced opening of Ca2+ channels and
uncoupling of endothelial nitric oxide synthase, followed by
platelet activation, adhesion, and aggregation, as evidenced by
the abundance of glycoprotein IIb/IIa and P-selectin on platelet
membrane surfaces. More importantly, these adverse effects on
platelets were found to be proportional to the particle
concentration, but inversely proportional to the particle
size.71 Furthermore, these effects could be explained by the
energy offset model proposed in ref 65 (Figure 4b). On the
basis of this model, the smaller silica nanospheres with a higher
external surface area per unit mass would be expected to release
more binding energy when internalized by platelet membrane,
and this energy compensates the bending energy cost
associated with the entrapment of the smaller particles by the
cell membranes. This, in turn, creates thermodynamically
favorable conditions for the platelet ingestion, activation, and
aggregation.
The effect of the size of MSNs on their distribution in vivo
was investigated in mouse models treated with MSNs having
different sizes. Shi et al. synthesized spherical MSNs 80, 120,
200, and 360 nm in diameter and traced their distribution and
fate in vivo after injecting the particles intravenously through
the mouse tail.74 Generally, the particles showed nontoxic or
noninflammatory effect to all the tissues in the mouse but
accumulated differently in them, in the order of liver > spleen >
lung > kidney/heart. Furthermore, the accumulation of MSNs
in each organ continuously increased in the first 5 days
postinjection but significantly decreased at 1 month.74
Moreover, higher concentration of the larger particles was
detected in urine samples 30 min after administration,
indicating that larger particles were easily captured by RES
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than the smaller ones.74 Conversely, MSNs with smaller sizes
circulated more in the blood for longer times without being
trapped in kidney.74 In another study, spherical MSNs with a
diameter of ∼100−130 nm were injected into mice at doses
ranging from 0.25 to 4 mg/mouse (i.e., ∼10 to 200 mg/kg
mouse body weight) once per day and continuously for 10
days.46 A higher concentration of liver transaminase was
detected in the mice treated with particles at a concentration of
2 mg/mouse or higher.46 Administration of up to 1 mg of
MSNs/mouse either by i.v. injection twice during a two-week
period or by i.p. injection twice per week for a period of 9
weeks caused no abnormality in mouse activity or organ
histology.46 These results suggested that the MSNs did not
induce acute and chronic toxicity. Elemental analysis of the
mouse urine and feces 1 day after the injection of MSNs
showed the presence of about 26.3% silicon in the mouse urine
but a negligible amount in the feces (Figure 5a). However, 2 to
4 days after injection of the mouse with MSNs, the
concentration of silicon (or silica) in feces kept increasing
but still remained significantly lower than that in urine. By day
4, the total excreted silicon (or silica) in the urine and feces
reached >94% of the amount originally injected into the mouse
(Figure 5a).46
The biodistribution of MSNs was further investigated by
administering fluorescently labeled MSNs in a mouse model
transplanted with human breast cancer cells that later
developed subcutaneous tumors.46 When the fluorescently
labeled MSNs were administered by i.v. injection, the particles
were found to preferentially bind to the tumor sites, possibly
because of the size match between the porous cavity formed in
the growing tumors and the nanoparticles circulating in the
blood.46 Furthermore, when the MSNs were loaded with the
anticancer drug camptothecin (CPT) and injected into the
mouse by i.p., they not only ended up more in the tumor but
also suppressed the tumor almost completely after 2 months. In
contrast, the CPT alone reduced the tumor down to only
∼14% of its original size, while the MSNs alone did not exhibit
any antitumor activity (Figure 5b).46 These results are clearly
due to the fact that the tumor blood vessels built around newly
grown cells are aberrant in their shapes from those around
normal vessels (Figure 5c).75 In other words, the tumor blood
vessels are leaky, thereby making the CPT-loaded MSNs enter
the cancer cells effectively.75 Furthermore, the result once again
indicates that pristine MSNs could be very promising drug
carriers for nanomaterial-promoted cancer therapy by taking
advantage of the enhanced permeability and retention (EPR)
effect in solid tumors. This characteristic cancer angiogenesis
particularly offers an opportunity for certain sizes of particles or
macromolecules (<200 nm in size), which can come with the
blood supply, to accumulate more in tumor vasculatures.76,77 As
MSNs can be easily designed and synthesized with optimized
sizes, they are conducive for use as drug-loaded nanocarriers to
successfully reach the tumor region and eliminate the tumor.
4.3. Effect of Particle Morphology. Particle morphology
is another important structural feature of MSNs that
determines their possible biological activities. Several different
studies on the effect of shapes of MSNs on biological systems
have also been conducted. To examine the effect of particle
morphology on internalization of MSNs into mammalian cells,
Lin and co-workers, for example, synthesized MSNs with two
different shapes and investigated their cellular uptakes by
Chinese hamster ovarian (CHO) and normal human fibroblast
cells.78 Specifically, the authors synthesized spherical shaped
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(80 to 150 nm in diameter) and rod shaped (400−1000 nm
length × 80−150 nm width) MSNs, possessing the same types
of fluorescent tags, similar surface charges (ξ-potential = −1.50
to −1.90 mv), similar surface areas (951.7 to 991.2 m2/g), and
similar pore diameters (∼2.7 nm). The two different types of
particles exhibited different transport properties to reach the
cytoplasm of the cells.78 Although all these different types of
particles got engulfed by the CHO cells more than by the
normal human fibroblast cells, the spherical ones were
internalized faster by both types of cells than the rod-shaped
nanoparticles, possibly due to the lower tendency of the former
to form aggregates.78 This result clearly demonstrates the effect
of shapes of MSNs on their biological activities. It is worth
noting that neither type of particle assaulted the nuclei of the
cells.78
As cellular membranes are the first physical barrier in cells for
nanoparticles to penetrate, MSNs with different morphologies
were expected to have different degrees of interactions with cell
membranes, and thus trigger different cascades of intracellular
events after entering the cells. This hypothesis was tested and
confirmed by several experimental works. For example, Huang
et al. showed different cellular uptakes and subsequent cellular
responses for MSNs with three different shapes.79 Although
MSNs with different aspect ratios, namely, 1:1, 2:1, and 4:1,
with dimensions ranging from 100 × 100 nm to 100 × 450 nm,
and possessing surface areas ranging from 791 to 1169 m2/g
were all found to be efficiently ingested by A375 human
melanoma cells via encapsulation within endosomes, those with
higher aspect ratios entered the cells more rapidly than those
with lower aspect ratios.79 Furthermore, the MSNs with higher
aspect ratios disrupted the cytoskeleton (F-actin disorganiza-
tion) of the cells and induced more cytotoxicity (in a dose-
dependent manner). Compared to untreated cells, the MSN-
treated cells also expressed less melanoma adhesion proteins.79
In particular, the particles with higher aspect ratios resulted in
much less protein expression, although they did not affect the
levels of mRNA concentrations,79 suggesting that some
damages to protein translation or post-translational modifica-
tion were dependent on the shapes of the MSNs.79 It is worth
noting that this result is slightly different from those reported in
refs 60 and 78; however, the latter were obtained with different
cell lines and nanomaterials. Thus, general conclusions about
the effect of shapes of nanomaterials on biological systems
cannot be easily made or at least require the consideration of
several other parameters including cell type and material
composition.
Our group also investigated the biological effects of two types
of MSNs possessing sizes in the same range but having different
shapes, i.e., spherical/oval shaped MCM-41 and irregular
shaped SBA-15, on suspended and adherent human cancer
cells.43 The results indicated that the MSNs had dose- and
time-dependent cytotoxicity in the first 0−2 days when
administered at concentrations in the range of 50−200 μg/
mL MSNs, with the MCM-41 MSNs exhibiting milder toxicity
to both types of cells than the SBA-15 MSNs.43 Furthermore,
the results indicated that both types of MSNs were efficiently
engulfed by the cells after 1 h of incubation time.43 Moreover,
our study on the impact of SBA-15 and MCM-41 MSNs on the
bioenergetics of the cancerous cells showed that only the
irregular shaped SBA-15 MSNs inhibited cellular respiration
and ATP formation of the cells (in a dose-dependent
manner).40 However, both types of MSNs were found to
impair oxygen consumptions of isolated mitochondria and
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submitochondrial particles, while neither particle was seen to
induce oxidative stress (i.e., the glutathione levels in both
MCM-41- and SBA-15-treated cells were not different from
that in untreated cells).40 Contrary to our results, Heikkilä et al.
reported that MCM-41 and SBA-15 microparticles (particle
sizes ranging from 1 to 160 μm) caused elevated apoptosis in
human colon carcinoma cells due to mitochondrial dysfunction,
which was promoted by the large ROS generated due to the
presence of the particles.47 The difference between the two
results might be attributed to the differences in particle
dimensions of the MSNs employed in the two studies, i.e., the
MSNs used in our case were ∼300−650 nm in diameter and
those investigated by Heikkilä et al. were 1 to 160 μm in
dimension.
In another study, we investigated the biological responses of
various extracted murine tissues, including lung, liver, kidney,
spleen, and pancreas, during exposures to 200 μg/mL calcined
SBA-15 and MCM-41 particles for hours.48 The results showed
that all the murine tissues did not exhibit any changes in their
microscopic structures and bioenergetics after exposure to both
types of MSNs.48 Further detailed in vitro studies with the lung
tissue from a mouse model showed that both particles were
widely distributed in pneumocytes, macrophages, endothelial
cells, fibroblasts, and interstitium, regardless of the particles’
shapes and sizes.80 These results clearly suggested that calcined
MSNs have very promising biocompatibility properties with
mammalian tissues when administered at reasonable dosages.
Similar examinations on the biocompatibility of MSNs, both
in vitro and in vivo, using MCM-41 (100−150 nm), SBA-15
(600−800 nm), and mesocellular foam (MCF, 4000−5000
nm) type MSNs at concentrations ranging from 100 to 500 μg/
mL were conducted by the Kohane group.39 These materials
exhibited in vitro toxicity at an incubation period of 4 days to a
variety of cells, independent of the type of the MSN particles
and their morphologies, in the following decreasing order:
human mesothelial cells > mouse myoblasts > mouse peritoneal
macrophases.39 More importantly, based on their in vivo data,
Kohane and co-workers suggested that the type of admin-
istration of the MSN particles strongly influenced how toxic the
nanoparticles would be. Whereas the administration of 30 mg
MSNs/mouse intraperitoneally and intravenously were proven
to be toxic, and even lethal in vivo, their subcutaneous injection
was harmless.39 This, in turn, suggested that the death of the
animal model treated with the MSNs might be due to
pulmonary embolism or thrombosis. Conversely, the result
implied that biodistribution of nanomaterials and their
subsequent toxicity, if any, could be altered by adopting
different routes of administration for them.76 In other words,
different administration methods of nanoparticles into bio-
logical systems could be used to help accumulation of the
nanoparticles at different desirable sites or targets.
Kohane and co-workers further pointed out that the
interactions between local tissues and MSNs could cause
significant systemic toxicity while the MSNs circulate in the
body.39 Hence, the fate of MSNs in the bloodstream deserves
more research in order to unravel the possible mechanism of
their possible toxicity in vivo and, more importantly, to
understand why some MSNs induce toxicity while others do
not.
Tang and co-workers also investigated the biodistribution
and excretion of different MSNs in mice, after injecting the tail
veins of mice with two rod-like fluorescein-conjugated MSNs
possessing different aspect ratios of 1.5 and 5 (or MSNs with
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Chemical Research in Toxicology
similar diameters but different lengths of ∼185 and ∼720 nm,
respectively).81 Two hours up to one day after injection,
stronger fluorescent signals were detected in the RES of the
murine liver, lung, and spleen, while weaker signals were
observed in the murine kidney. Whereas the MSNs were found
in both urine and feces samples, they were detected in lower
amounts in the kidney than in the liver, suggesting that the
MSNs could be more rapidly excreted via renal uptake than
hepatic digestion,81 a result which was consistent with that
reported in ref 46. The fluorescent signals of these MSNs
started to fade away seven days after administration as a result
of degradation or excretion of the particles. Meanwhile, no
fluorescent signals were observed in limb lymph nodes and the
brain, suggesting the inability of MSNs to cross some physical
barriers such as the blood−brain barrier.81 Furthermore, 2−24
h after administration, the concentration of the MSNs with the
larger aspect ratios remained almost unchanged in the
bloodstream, whereas the concentration of the MSNs with
the smaller aspect ratios dramatically decreased,81 indicating
that the higher aspect ratio rod-shaped MSNs circulated for
longer periods in the bloodstream than the shorter ones.
Hence, it can be concluded that the shape (or aspect ratio) of
MSNs can also influence the MSNs’ circulation times in the
bloodstream and possibly their interactions with biological
systems as well.
4.4. Effect of Mesoporosity and Pore Sizes. The
mesoporous structures and the so-caused high surface areas
are characteristic features of MSNs that can also impact the
materials’ biological activities. This is well demonstrated by
some comparative biological studies involving MSNs, and as
reference materials, some dense silica nanoparticles that possess
chemical composition similar to that of MSNs but have much
less surface area than MSNs were used. For instance, whereas
MSNs catalyzed the oxidation of epinephrine, a biogenic
hormone, in phosphorated buffer solution due to the possible
generation of oxygen radicals inside the mesoporous channels
of MSNs, the dense silica nanoparticles did not catalyze this
oxidation reaction.82 Furthermore, when incubated with
cancerous cells, the solid silica spheres exerted a more acute
and permanent injury to cells and induced more cell death than
MSNs. This indicates that the dense silica nanospheres were
more severely cytotoxic, or conversely, the MSNs were more
biocompatible. This is presumably due to the presence of
mesoporous structure in the latter or the absence of
mesoporosity in the former, although other subtle structural
differences between the two types of materials might have also
led to the differences in their biological effects.43
Two independent groups also showed that MSNs exhibited
less hemolytic activity compared with their dense silica
counterparts possessing sizes similar to those of the MSNs
but no mesoporous structures in them.64,83 The authors
attributed this difference in the hemolytic effect exhibited by
these two materials to the differences in their surface silanol
density and overall cell-contactable surface areas. MSNs with
fewer silanol groups on their cell-contactable surfaces were
considered to trigger the hemolysis of RBCs less than their
nonporous silica counterparts containing higher density of cell-
contactable surface silanol groups.64
As introduced above, MSNs can be synthesized with different
mesostructures such as hexagonal, cubic, worm-hole, etc.
Besides sizes and shapes, the type of mesostructures existing
in MSNs was also found to affect the MSNs’ biological activities
as illustrated with the following examples.
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The first example deals with MSNs' catalytic activity toward
the oxidation of epinephrine. As mentioned above, both MCM-
41 and SBA-15 MSNs were shown to catalyze the oxidation of
epinephrine; however, the two materials exhibited different
catalytic activities toward this reaction, which was attributed
mainly to their differences in mesostructures.82
In another example, whereas SBA-15 type MSNs impaired
cellular respiration and mitochondrial electron transport chain
in cells when incubated in vitro with Jurkat cells, MCM-41 type
MSNs at the same dosage barely resulted in any noticeable
effect.40 In addition, the MCM-41 type MSNs generally
produced milder toxicity than SBA-15 type MSNs.43 Although
these two types of MSNs have similar hexagonally ordered
mesostructures, they have some subtle differences. For instance,
in contrast to MCM-41, SBA-15 has a unique porosity as it it
possesses pore interconnectivity between the ordered cylinder-
ical channel pores; this in turn, contributes to a substantial part
of total surface area and leads to a different catalytic activity
from MCM-41.82 Thus, some different biological effects
exhibited by these two types of MSNs can be explained
based on these structural differences. The relatively larger
surface area and smaller pore diameter of MCM-41 can make
these nanoparticles thermodynamically more favorable for
cellular ingestion, according to the proposed model regarding
nanoparticle endocytosis shown in Figure 4b.65 Our previous
study also indicated that the MCM-41 and SBA-15 underwent
cellular internalization through slightly different endocytotic
mechanisms, as shown in Figure 6a.43
In an additional example, MSNs having different meso-
structures (short-range worm-like or long-order hexagonal
structure) were reported to rupture RBCs differently.64 This
finding further confirms that a defined mesoporous structure,
including pore size, pore volume, degree of mesostructure
order, and integrity, can determine how MSNs interact with
biological systems. Thus, like the particle size, morphology, and
surface area, the type of mesostructure in MSNs should not be
neglected as far as the biological responses of MSNs are
concerned. The difference in mesoporosity of the MSN
materials can also result in different drug adsorption capacities
and drug release profiles, which in turn leads to different
pharmacokinetics when the drugs are delivered by different
MSNs as drug carriers. All these results point out the
significance of the mesoporous structures of MSNs to their
biocompatibility as well as bioactivity in various biological
systems.
4.5. Effect of Surface Property. Given the interests in the
potential biological applications of MSNs, synthetic methods
that make MSNs as biocompatible as possible are necessary. In
this regard, surface modification of MSNs is very important as it
may result in enhanced cellular uptake and endosomal escape,
and reduced collisions with some undesired organelles during
cellular trafficking of the nanoparticles. This can be exemplified
by the work performed by Lin and co-workers, in which a
variety of surface-functionalized MSNs were found to be
internalized differently into human cancerous HeLa cells
expressing folate receptors.84 The different surface-modified
MSNs were synthesized by functionalizing MCM-41 with 3-
aminopropyl (AP), guanidinopropyl (GP), 3-[N-(2-
guanidinoethyl)guanidine]propyl (GEGP), and N-folate-3-
aminopropyl (FAP) groups, All these organic-functionalized
MSNs exhibited lower surface areas and pore volumes
compared with their parent MCM-41 in the order of parent
MCM-41 > AP-MCM-41 > GP-MCM-41> GEGP-MCM-41 >
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Figure 6. (a) TEM images showing the endocytosis of MCM-41 and
SBA-15 MSNs by Jurkat cells.43 Reprinted from ref 43. Copyright
2009 American Chemical Society. (b) Endocytotic pathway of MSNs
in mammalian cells.
FAP-MCM-41.84 Interestingly, the degree of particle uptake by
the HeLa cells increased in the same trend as their surface
charges, i.e., in the order of parent MCM-41 < AP-MCM-41 <
GP-MCM-41 < 0 mv < GEGP-MCM-41 < FAP-MCM-41.84
Furthermore, among these materials, the FAP-modified MSNs
were internalized the most by the folate receptor-expressing
cells, indicating that chemical affinity of functionalized particles
to cells could play greater roles than physical properties in
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determining the degree of endocytosis of the MSNs. The
mechanisms for the endocytosis of the particles, except for
GEGP-functionalized MSNs, were also included in the study,
and they were reported to be different from material to
material.84 The authors concluded that the parent MCM-41
MSNs were ingested through a clathrin-mediated pathway,
whereas the AP- and GP-grafted MCM-41 MSNs were
internalized into cells via a caveolae-dependent mechanism,
and the FAP-MCM-41 MSNs were engulfed by both clathrin-
and foliate receptor-mediated endocytosis, followed by more
confinement of positively charged particles inside endosomes.84
These results clearly showed the effects of surface properties,
such as surface electrochemistry, of MSNs on their cellular
uptake and subsequent endosomal escape. Notably, a particular
surface chemistry, surface charge, or surface functional group
may help one of the steps in the given biological process, but
may also inhibit the others in the process. Therefore, all the
steps in a given biological process, e.g., cellular uptake, need to
be taken into account when rationally designing and synthesiz-
ing nanomaterials for biological applications.
Beginning with cellular uptake of the foreign nanomaterials,
the consequent cellular events can depend on the various
physical and chemical factors of the nanomaterials, such as their
sizes, surface charges, surface ligands, etc. Hence, evaluations on
the combined effects of all the material variables in a series of
the biological processes, which consist of membrane trespass-
ing, vesicular coating, endosome development, and lysosome
degradation, would be necessary for one to determine how
exactly the cellular trafficking of MSNs occurs. We emphasize
here that the surface chemistry of MSNs, including their surface
charges and ligand affinity, needs to also be assessed when the
biological responses of MSNs are investigated or the potential
biomedical applications of MSNs are evaluated. For example,
we previously found that quaternary amine-functionalized
MSNs (positively charged) tended to stick onto negatively
charged cell membranes of the cells, rather than penetrating the
cells and entering the cytoplasm.43
Generally, the uptake of nanosized particles into cells can be
a complicated process, typified by phagocytosis or different
receptor-mediated endocytosis (shown in Figure 6b). In
eukaryotic cells, regardless of each specific pathway, endocytosis
usually takes place when the plasma membrane (inner cellular
membrane comprising glycolipids and integral proteins and
covering the cytoskeleton) forms vesicles to wrap the particles
and generate early endosomes. This process is reversible as
endosome-transported particles can be repatriated out of the
cells, and endosomes are recycled to compensate the plasma
membrane. Alternatively, early endosomes mature into late-
stage endosomes with increasing acidity due to the elevated
amount of acid hydrolases.85 The matured endosomes then
fuse with lysosomes, which are mainly produced by the Golgi
apparatus and endoplasmic reticulum.85 The lysosomes, which
are full of hydrolytic enzymes, then help the degradation of the
particles. The nondegradable particles are eventually exocy-
tosed.85
Thus, in order to become effective in therapeutic or
diagnostic applications, MSNs need to be well designed so
that they are capable of crossing cell membranes, fleeing
endosome capture, dodging unwanted intracellular binding
processes, and finally reaching their cellular targets and
releasing their payloads of drugs, biomolecules, and bioactive
agents, as necessary. Among these processes, the endosomal
escape of MSNs constitutes a crucial step or checkpoint, which
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Chemical Research in Toxicology
can be significantly improved by properly functionalizing the
surfaces of the MSNs. For example, polycations such as poly(L-
lysine), poly(ethyleneimine) (PEI), polyamidoamine den-
drimers, natural chitosan, and endosome-disrupting peptide
were successfully used to coat the outer surfaces of MSNs in
order to improve cellular uptake and endosomal release of
MSNs.86 Furthermore, as branched PEI contains a high density
of amine groups, it can act as a proton sponge and thus serve as
an excellent proton buffering medium during endosomal
encapsulation, where pH values abruptly decline (from 7.4 to
∼5.5 in the late endosome).87 Therefore, due to this high
endosomolytic activity, PEI-coated MSNs exhibit an improved
ability to deliver nucleotides and drugs to intracellular or even
nuclear sites.88
Similarly, chitosans, poly(alkyl acrylic acids), and anionic
amphiphilic peptides (derived from viral fusion peptides),
which get protonated in response to pH decrease, can also be
tethered on MSNs’ surfaces via covalent bonding or electro-
static interaction. This functionalization can help MSNs
accumulate with high charge density when acidity increases
during endosome development, destabilizing the endosomes’
membranes.86 In addition, surface modification of MSNs with
functional groups such as photosensitive porphyrins was shown
to result in a novel MSN-based delivery system for photo-
induced endosomal escape in living cells.89 After entering cells
via endocytosis and binding to endosomal membranes, these
photosensitized MSNs were excited by light and further
quenched by a triplet oxygen moiety to produce a singlet
oxygen species.89 The singlet oxygen species then damaged the
endosomal membranes, thus releasing the cargo-loaded MSNs.
Tuning the surface chemistry of MSNs is imperative also for
potential in vivo administration or future clinical uses of MSNs
as drug delivery vehicles or bioimaging agents. Surface
modification can help MSNs camouflage, and this process
assists the MSNs to avoid massive opsonization and be rapidly
eliminated by RES of the body’s immune system. Polyethylene
glycol (PEG) has been widely utilized for these purposes as it
has the ability to improve the surface hydrophilicity of MSNs,
reduce their protein adsorption, and shield them from
nonspecific bindings.90 Compared to their naked counterpart,
PEGylated nanoparticles (including PEGylated MSNs) were
already shown to have much higher dispersion and stability
(resistance to degradation by aging) in biological media and
much lower adsorption of serum proteins and less uptake by
macrophages.90−92 PEGylated MSNs with enhanced biocom-
patibility could be produced more easily by applying PEGs with
molecular weights >10 kDa on the nanoparticles and by
ensuring homogeneous surface coverage and optimal PEG
density on the nanoparticles’ surfaces rather than by increasing
their chain lengths.90−92 In addition, properly performed
PEGylation could also help the mesoporous structures of
MSNs remain intact.21,64,65,92 Furthermore, PEGylated MSNs
were shown to exhibit significantly lower effects toward
hemolysis of human RBCs, possibly because the PEG groups
shielded the silanols on the surfaces of MSNs and prevented
the latter from possibly generating ROS that could damage cell
membranes. This was confirmed both with in vivo and ex vivo
mouse model studies in which the distributions of PEGylated
MSNs in various tissues following i.v. injection were probed.
PEGylated nanoparticles also exhibited longer retention times
in the bloodstream and lower accumulation in the spleen, liver,
kidney, and urinary bladder, compared to their parent MSNs or
their carboxylated or hydroxylated counterparts with similar
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particle sizes.74,81,92,93 In addition, PEGylation prolongs the
blood circulation of MSNs by reducing RES organ uptake,
bypassing renal and hepatobiliary clearance, and thus enhancing
permeability and retention of the nanoparticles. Hence, the
surface modification of MSNs using PEG or other similar
functional groups not only improves the biocompatibility of
MSNs but also promotes the ability of the MSNs to deliver
drugs or biomolecules to desired sites in a sustained manner.
The surface property of nanomaterials can also affect how
nanoparticles undergo exocytosis as much as it affects their
endocytosis and intracellular trafficking. However, unlike
endocytosis, which is more often looked into while studying
the biocompatibility of nanomaterials, exocytosis of non-
degraded nanoparticles such as MSNs has been largely
overlooked. Generally, exocytosis is a cellular process by
which cells remove the unwanted material or wastes from the
plasma membrane typically via the following processes: (1)
intracellular vesicles containing the material to be disposed are
transported to the vicinity of the inner plasma membrane; (2)
once the intracellular vessicles are in contact with the plasma
membranes, the vesicles are further fused and integrated onto
cell membranes; and (3) with vesicular membranes turning
themselves inside-out, the waste to be disposed is wrapped and
released out of the cell.85
Exocytosis of nanomaterials is an inevitable step that
determines the final fate of the nanoparticles used as drug
delivery vehicles or hosts for bioactive reagents. Furthermore, it
determines how compatible the nanoparticles are with the
biological systems. Therefore, exocytosis also requires attention
during investigations of the overall effects of nanoparticles on
biological systems. Not surprisingly, like endocytotic processes,
exocytosis can be influenced by many physiochemical variables
associated with the nanomaterials such as size, shape, surface
property, etc. and the biochemical or biological factors of host
cell/tissue, such as cell/tissue origins, protein expression, and
ion strength of the intracellular environment.
Although only a few studies have been conducted on
exocytosis, there have been some reports on exocytosis of
MSNs in different cell lines. For example, Slowing et al. studied
the exocytosis of MSNs by normal (HUVEC) and cancerous
(HeLa) cells and reported two interesting findings.94 First, the
MSNs were found to be ingested by the cells and reached a
constant intracellular amount within 2 h, indicating the
attainment of a balance between the rates of endocytosis and
exocytosis of the particles (or equilibrium) in 2 h of incubation
time. Second, the exocytosis as well as transcytosis (a process in
which materials are transported from one to another cell) of
MSNs were found to be much more efficient in healthy
HUVEC cells than in malignant HeLa cells.94
Despite these exocytosis studies, systematic in vitro studies
on the effect of size of MSNs on their exocytosis are, however,
limited. On the contrary, such studies have been conducted for
many other materials including silica spheres (60−600 nm in
diameter), gold nanoparticles (10 nm in size), or metal
quantum dots (4 nm radius), which generally showed higher
exocytotic efficiency for the smaller nanoparticles.95−97 Never-
theless, the effect of surface charge on secretion of the
nanoparticles in murine models was well investigated for
MSNs; the results showed that the surface charge on MSNs
plays an important role in regulating the cellular excretion of
MSNs.98,99 Specifically, the study showed that MSNs, especially
those with sizes of >100 nm, were primarily retained by the
liver and to a lesser extent by the spleen after being
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Chemical Research in Toxicology
administered via tail-vein injection in nude mice and Sprague−
Dawley rats.98 Compared with their negatively charged
counterparts, the positively charged MSNs at physiological
pHs were more adsorbed by serum proteins, thus being more
rapidly transported via hepatobilic secretion into the gastro-
intestinal tract and finally eliminated with feces.98 When hepatic
metabolism of MSNs was further investigated at subcellular
resolution in real-time, the results revealed that the negatively
charged MSNs had a tendency to remain in blood vessels or be
sequestered by Kupffer cells that constitute the walls of liver
sinusoids.99 As a result, negatively charged MSNs could be a
potential threat to hepatic health. On the contrary, after
efficiently endocytosized by hepatocytes, the positively charged
MSNs were effectively eliminated via hepatobiliary pathway.99
These results not only showed the effect of surface charges of
MSNs on cellular uptake and host clearance of the nano-
particles but also depicted charge-dependent surface recog-
nition and disposal routes by different cells/tissues.
4.6. Effect of Cell Type. As mentioned in some of the
examples in the above section, the biocompatibility of MSNs
could be strongly dependent on the type of cell- or tissue/organ
in question.
Thus, to properly evaluate the biocompatibility of engineered
nanoparticles such as MSNs, an appropriate biological system
needs to be wisely chosen, and the evaluation should be
accompanied by accurate, reliable, and expedient assessment
methods. Generally, compared to the more complicated in vivo
models, in vitro studies present much quicker, relatively clearer,
and more specific results for screening the biocompatibility of a
new material while also minimizing animal sacrifices. Different
in vivo and in vitro studies on the interactions between various
nanomaterials (e.g., carbon nanotubes and quantum dots) and
diverse biological entities (animal/plant cells, microorganisms,
tumor, or transgenetic models, etc.) have been widely
documented in the literature.1−6 However, here we mainly
focus on those studies adopting in vitro systems because most
of the reports in this area have dealt with such systems.
Furthermore, the discussion here will mainly illustrate the
possible interference of the cell types on toxicity assessments.
It is demonstrated with some examples above that the
potential cytotoxicity (or biocompatibility) of nanomaterials is
dependent on the cell type in question. As an additional
example, it is worth mentioning the work by Chang et al. on the
effect of dense nanosized silica particles on a variety of adherent
cancerous (lung, gastric, and colon epithelial cancers) and
normal (skin and lung fibroblast) cells.100 The authors found
that the cytotoxicity of the silica nanoparticles was strongly
correlated with the types of the cells employed in the study,
where different cell types exhibited different metabolic activities
and doubling times with the nanoparticles. Overall, the
nanosized silica particles induced lower cytotoxicity, and
thereby shorter cell doubling times, on the cells having higher
metabolic activity.100 Our research with MCM-41, SBA-15, and
solid spherical nanosized silica particles, with and without
surface amine groups, also indicated that the particles exhibited
different toxicities on different cell lines.43 In contrast to Jurkat
(human T-cell lymphoma) cells grown in suspension media,
adherent SK-N-SH cells derived from human neuroblastoma
showed more drug resistance and less recovery when treated
with all the three types of nanomaterials, i.e., MCM-41, SBA-15,
and solid spherical nanosized silica particles, whether they were
modified with amine groups or not. This difference appeared to
be mainly due to the possible differences in endocytosis and
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exocytosis of these materials in the two cell lines.43 Therefore,
in addition to the cellular metabolism, the growth pattern of
cells (i.e., whether being adherent or suspended cells) can also
determine how the cellular responses during exposure to MSNs
would be.
By using hydroxyapatite nanoparticles with similar particle
size, shape, and crystallinity but different surface charges, Chen
et al. recently showed that these differently charged nano-
particles underwent different cellular uptake in murine
osteoblasts in vitro.101 The particles with positively charged
surfaces were accumulated inside the cells more than their
negatively charged or neutral counterparts. This result is
consistent with a previous report on the uptake of differently
charged MSNs by HeLa cells.84 Interestingly, the cells even
with highest particle uptake (i.e., those containing positively
charged hydroxyapatite nanoparticles) had cell membrane
integrity, viability, and normal replication, with no difference
compared to those containing less particles inside (i.e., the cells
treated with negatively charged or neutral particles).101 This
result was unprecedented, considering the fact that substantial
residues of nanomaterials remained inside the cells or tissues
and that such materials are traditionally expected to result in
severe injury or toxicity based on previous findings on the
internalizations of hydroxyapatite nanoparticles in other cells
(e.g., fibroblasts or hepatic cancer cells) and their cytotoxicity
and cell growth inhibitory effect.101 Nonetheless, this again
corroborated the results that surface charge on MSNs could
play a major role on cell uptake of the materials and cell
viability. Moreover, the results showed that the degree of cell
uptake of the functionalized MSNs and their subsequent
biological effects and biocompatibility could vary with the cell
type used in the study.
In the work by Chung et al., a similar observation on the
effect of surface charges of MSNs on the biological activities of
these materials on human stem cells was also reported.102
Specifically, the authors adjusted the density of positive charges
on the surfaces of MSNs by varying the relative density of their
surface amine groups and then monitored how well the
resulting particles were engulfed by the cells. Whereas the
MSNs were effectively internalized by human mesenchymal
stem cells regardless of the nanoparticles’ surface charges, the
more positively charged MSNs were found to be more ingested
in mouse embryonic cells, without affecting cell viability,
division, and differentiation.102 These results clearly indicate
that different endocytotic mechanisms must be at play during
internalization of a given MSN in different cell types.
Therefore, the biocompatibility of a given nanomaterial or
MSN can vary depending on what type of cell the materials are
with. For this reason, one may have to also expect that the
nanomaterials administrated as nanomedicines could interact
differently with different targets, such as tumors with different
origins, sizes, growth rates, biochemical markers, etc. As a
result, different therapeutic or diagnostic effects can be
anticipated by a given nanomaterial-based drug delivery vehicle
at different parts of the body.
4.7. Effect of Methodology of Toxicity Assessment.
Generally, the assessment employed in a given study could
affect the experimental outcomes and the final conclusions one
could make with regard to the possible biocompatibility of a
certain nanomaterial on the given cell line. Thus, it is essential
to discuss the toxicity assays that different researchers have
frequently used, which might also be responsible for some
contradictory results reported (or possibly will be reported in
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Chemical Research in Toxicology
the future) on the toxicity (or biocompatibility) of MSNs in
different studies.
For example, an important study conducted by Krug and co-
workers in 2006 revealed many possible sources of artifacts in
cell viability tests, which led to contradictory results about the
toxicity (or biocompatibility) of nanosized materials.103 For
instance, when human A549 lung epithelial cells were treated
with single-walled carbon nanotubes (SWCNTs), the tradi-
tional MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide indicated that SWCNTs caused
severe cell injury, whereas other standard tests suggested
otherwise.103 This underscores how the information reported
about the biocompatibility of nanomaterials relies also on the
methods employed to assess them. Thus, the underlying results
from these tests may have to be taken with caution. This also
applies to MSNs when their biocompatibility is under
examination.
Some possible sources of artifacts and errors during
biocompatibility tests might be related to how the assays
work or the inherent processes associated with them. MTT or
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
is a yellow salt that can be reduced by mitochondrial
dehydrogenase in living cells to produce a water-insoluble
purple formazan salt intracellularly. After the cells are
homogenized and the salt is solubilized (e.g., by dimethyl
sulfoxide), the formazan product can be quantitated by
spectrometrically monitoring the intensity of the absorption
maxima at ∼570 nm, which in turn gives information about the
number of living cells (or cell viability).104 This easy-to-use
assay allows the rapid analysis of cell growth and proliferation,
particularly in drug screening and cytotoxin evaluation;
however, the possible cytotoxicity of MTT itself in different
cell lines remains a question. Furthermore, during toxicity tests
with MTT, the formazans sometimes form insoluble crystals,
which could not be further removed by a solubilization reagent,
inside porous nanomaterials such as carbon nanotubes.103 This
might, in turn, cause significant decrease in the color or
absorbance of the solutions and underestimate values of cell
viability. MTT was also found to be an inefficient reagent for
examination of the potential toxicity of mesoporous silicon
microparticles because it oxidized the surface of silicon into
silica,105 thus leading to overestimated viability compared to
that expected for such materials. Although silica cannot be
further oxidized and does not interact much with MTT, cell-
metabolized formazan products were visualized on cellular
surface after a half-hour treatment of the cells with MSNs,
indicating the presence of enhanced formazan exocytosis due to
MSNs, which in turn led to an overestimation of the MSNs’
cytotoxicity.106 Moreover, as surface functionalization did not
alleviate this effect, the MTT assay was shown to be somewhat
inappropriate for assessing the toxicity of MSNs.106
Later on, different tatrazolium salts that can produce water-
soluble formazans, including XTT (2,3-bis-(2-methoxy-4-nitro-
5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymehoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium), WST series 1−11 (water-soluble
tetrazolium salts developed by Dojindo Laboratory, Japan),
and INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5phenyl-2H-tet-
razolium chloride) emerged as possible replacement reagents
for MTT. These reagents require no solubilization and render
much improved signals. Thus, these nontoxic tatrazolium
compounds and their corresponding water-soluble formazans
are more suitable and effective for more precise determination
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of the cytotoxicity of nanosized porous materials, including
MSNs.41,43,103,106,107
High-throughput screening of the toxicity of nanomaterials
using multiwall cell plates for adherent cells has been widely
investigated in recent years. However, care needs to be
exercised while using this method, particularly when differ-
entiating the cytotoxicity inherently caused by the nanoparticles
from that caused by the possible physical blockage of oxygen or
nutrients to the cell surfaces by the nanoparticles.39,43 There is
one solution to this potential problem, which is achieved by
putting the particles into the multiwall cell plates first, followed
by adding the cells into the solution, and then determining the
biological responses of the cells to the particles.39 The toxicity
data obtained with this approach can then be compared with
those obtained by performing the experiment in a reverse order,
that is, growing the cells in the wells first, followed by adding
the particles, and then measuring the biological effects of the
particles. However, concerns about whether the initial coverage
of MSNs on the bottom of cell plates would influence the cell
seeding should be also taken into consideration, as a significant
amount of MSNs might occupy the cell growth area and affect
cell attachment to the cell plates.
In addition, sedimentation of insoluble particles by gravity
from their suspension to land onto adherent cells and contact
the cell surface remains a major issue in many cytotoxicity
tests.108 This is because, without the particles virtually
contacting the cells, their possible effect on the cells cannot
be properly studied. This is especially more serious for MSNs
with small sizes, low densities, and large surface areas per unit
mass, and MSNs possessing surface charges. However, other
forces (e.g., convection force) would help such nanomaterials
contact the cells more than gravitational force does.109 In any
case, the reported dosage and incubation time that cause cell
mortality could be overestimated if the experiments are
conducted without taking these issues into consideration.
Moreover, realistic time periods required for the nano-
particles to diffuse and reach cells should be subtracted from
the typically reported incubation times in most studies, which
usually start right after the addition of the nanoparticle
suspensions into cell media. Therefore, during days-long cell
incubation, the real portion of particles to reach surface layers
of the cells at the bottom of the wells, where adherent cells
reside, could be very small, invalidating any possible non- or
low-toxicity conclusion about certain nanoparticles, including
MSNs of certain sizes with high surface areas and low density.
Furthermore, to make matters worse, some small particles can
form aggregates and rapidly sediment onto cell surfaces
differently under different conditions.109 Thus, materials (e.g.,
MSNs) should be sonicated until a well-suspended solution is
obtained, and then the cell plates after the addition of
nanomaterials should be gently and briefly centrifuged to
promote the incubation of cells with the dispersed nano-
particles.
A possible source for the evident toxicity induced by the
treatment of MSNs comes from the possible residual organic
templating reagents in the MSNs. In addition, due to the
different synthetic procedures and postsynthetic processes,
subtle differences in the density of surface silanol groups in the
MSNs can also sway the final conclusion on their
biocompatibility with certain biological objects, such as RBCs.
Thankfully, many kinds of very good characterization tools,
ranging from solid state NMR spectroscopy and elemental
analysis to thermogravimetric analysis and infrared spectrom-
dx.doi.org/10.1021/tx300166u | Chem. Res. Toxicol. 2012, 25, 2265−2284
Chemical Research in Toxicology
etry, are now widely available to allow a routine and reliable
determination of the structural as well as compositional
parameters associated with these nanomaterials. These help
to easily rule out the possible toxicity due to the detrimental
effects of residual organic templating agents in MSNs after the
preparation of the MSNs.
In toxicity assessments, colorimetry is one of the most
commonly used methods to conveniently elucidate the
biological effects of nanomaterials and to quickly determine
their general cytotoxic profiles.110−113 Besides colorimetric
methods, other conventional measurements that enable the
detection of ROS generation or DNA damage, such as flow
cytometry, high-performance liquid chromatography (HPLC)
or immunostaining, have also been utilized to evaluate the
cytotoxicity of MSNs and unravel the possible mechanism
behind their cytotoxicity, if any.110−116 As there are no reports
regarding the severe interference of nanomaterials in these
analytical methods, these techniques could be reliably used to
assess cellular injury induced by MSNs. Nonetheless, to fully
probe and verify the biocompatibility of well-defined nano-
scaled materials, including MSNs, it is still highly recommended
that many complementary methods as well as more than one
type of cell line should be employed.
5. CONCLUSIONS AND PERSPECTIVES
In this review, we first introduced the various synthetic
strategies used to make and functionalize different types of
MSNs. We then discussed how the physiochemical properties
of MSNs could be tuned, either by shape-/size-controlled
synthesis or by functionalization of their surfaces with organic
groups. Furthermore, we described how the surface function-
alization and shape/size control of MSNs could, in turn, result
in MSNs with versatile properties for various potential
biological and medical applications.
The review also highlighted many of MSNs’ unique structural
features such as nanosize pores, mesostructures, high surface
area, high pore volume, and easily modifiable surfaces and
elaborated on how these structural features enabled MSNs to
become a very promising class of candidate materials for drug
delivery, host materials for bioimaging agents, and platform
materials for biosensing/biocatalytic moieties. However, it was
also shown that many different structural features of MSNs
could lead to different biological activities for these materials.
Thus, probing and summarizing the overall biological responses
induced by these materials, which possess a range of structural
features and compositions, could be challenging. Nevertheless,
in light of their great potential applications in biomedical areas,
many experts in the field seem to agree that the biological
activities of these materials need to be fully assessed and
evaluated in order to determine the proper physical or chemical
traits that could make MSNs highly biocompatible. These
efforts and some typical results reported in these areas over the
last two decades have been discussed as the main subject in this
review.
A broad evaluation at the interface between the material’s
properties and its biological surroundings is crucial to fully
determine whether a given material is biocompatible. This task,
however, is very complicated and challenging in the case of
engineered nanomaterials such as MSNs because they possess a
variety of physical properties and chemical compositions, which
could lead to a diverse range of biological responses and trigger
a cascade of different biological events. While intense studies
were being conducted by various researchers worldwide to fully
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understand these structure-related biological effects of MSNs,
including their possible negative impacts on human health,
some of the studies also revealed contradictory results about
the biocompatibility (or cytotoxicity) of various MSNs, as we
discussed in this review.
The chemical modification, along with the original synthetic
condition, can tailor the particle size, shape, or surface property
of the MSNs. This, in turn, could influence the biological
activities of the nanomaterials. This aspect about functionalized
MSNs was also considered in this review. In particular, it is
illustrated that the favorable size, shape, or surface property of
organic-functionalized and nonfunctionalized MSNs alike that
leads to insignificant interference in a given biological system
can generally be attributed to those that cost the least interfacial
energy at the nanobio boundary. Specifically, in contact with
cell or intracellular organelles, MSNs of a particular size and
shape with larger surface area can be thermodynamically
favored in their cellular interactions, leading to more intra-
cellular accumulation but less cell injury. Thus, proper surface
modification of MSNs (e.g., making the MSNs with positively
charged surfaces) can enhance their endocytosis as well as
exocytosis, while also mollifying their intracellular trafficking.
However, size may play a superior role over shape in causing
toxicity in vivo, if any, as the body generally recognizes a certain
dimension of foreign particles, allowing or disallowing their
further penetration into different organs or sites in the body to
take part in some biological processes. At the same time,
different surface properties regulate the particle circulation in
the bloodstream, whereas the different affinities to certain
biological receptors due to functionalization could dictate the
final fate or biocompatibility of the particles.
In this review, we also highlighted how the dosage of MSNs
affect the biological responses to MSNs, and why their dosage
should be properly chosen when they are considered for
biological applications to minimize or eliminate their unwanted
side effects (e.g., toxicity). In addition, the different chemical
functional groups that can be immobilized on the surfaces of
MSNs via surface modification to improve the selectivity and
the affinity of MSNs to bind to specific biological sites were
discussed.
In our opinion, mesoporosity is among the main structural
features of MSNs that also strongly affects the biological
activities of these materials, besides the size, shape, and dosage
of MSNs. This structural character also substantially determines
whether MSNs can serve important functions in biological/
medical applications, such as drug delivery vehicles. Compared
to the solid nature of other silica nanospheres, the
mesoporosity of MSNs is responsible for their lower rigidity,
buffering their interactions with biological entities. The
mesoporous structure is also considered to be the major
reason for the lowered interfacial energy between these
nanomaterials and biological surfaces. Such structure-related
biological features of MSNs have also been discussed in this
review.
For a given MSN, it was clearly shown that different
biological systems are expected to exhibit different responses.
At this point, it is worth noting that the conclusions made
about the biocompatibility of a given nanomaterial, including
MSN, are strongly determined by the accuracy or reliability of
the chosen biological assay. Moreover, when using traditional
assays to probe the biological effects induced by nanoscaled
materials, the possible interruption in data collection or analysis
during the assessment remains a big concern, which may result
dx.doi.org/10.1021/tx300166u | Chem. Res. Toxicol. 2012, 25, 2265−2284
Chemical Research in Toxicology
in artifacts or lead to wrong conclusions. This issue has long
been overlooked in many studies and may be a substantial
reason (at least, partially) for many conflicting results reported
for a given nanomaterial (e.g., MSN) by different research
groups, who have employed different experimental methods for
their studies. Thus, possible pitfalls in those traditional assays
used to determine the biocompatibility of MSN were also
analyzed in the review.
The investigation of the overall biocompatibility of the
nanomaterials, including MSNs, can be complicated by the fact
that the nanomaterials produced with typical nanomaterial
synthesis often have a range of physicochemical properties and
some size distribution, among other things. Thus, in our
opinion, it is highly important and critical that the biological
research involving MSNs should start with finding or
developing reliable synthetic routes to a set of nanoparticles
with well-defined structures. This has to be followed by
choosing reliable and appropriate biological systems to conduct
the biological studies on biocompatibility. Sometimes, more
than one type of reliable assay (based on different distinguished
and complementary analytical methods) is also needed to
properly determine the overall biocompatibility of the nano-
materials within a defined biological system. With an
accumulated knowledge on the effects of a certain nanoma-
terial, such as MSN, under a range of biological conditions, a
general biocompatibility of such a material can thus eventually
emerge. For example, complete information about the exact
cutoff size of MSNs that results in biocompatibility or causes
cytotoxicity, information that is still missing in the literature but
is extremely important to move the field forward, can be
obtained. Another important immediate research in this area
has to be determining the exact effect of surface silanol groups
and surface morphology on the interaction of MSNs with
biological systems. With the recent advent of more powerful
atomic force microscopes (AFMs) and their successful
applicability to soft materials, this type of study should now
be more feasible to conduct. This type of study, in turn, could
provide an enormous amount of information regarding MSN−
bio interfaces, MSNs’ interactions with biological systems, and
why some MSNs could be biocompatible while others could be
toxic.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: tasefa@rci.rutgers.edu (T.A.); ztao@tsinghua.edu.cn
(Z.T.).
Funding
This work was supported by Tsinghua Research Fund
100401007, Chinese Postdoctoral Science Foundation Grant
20100480314 (awarded to Z.T.), and the US National Science
Foundation (NSF) under Grant Numbers CAREER CHE-
1004218, NSF DMR-0968937, NSF NanoEHS-1134289, NSF-
ACIF for 2010, and NSF Special Creativity grant in 2011
(awarded to T.A.).
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS
AP, 3-aminopropyl; CHO, Chinese hamster ovarian; CPT,
camptothecin; DCs, dendritic cells; EPR, enhanced perme-
ability and retention; FAP, N-folate-3-aminopropyl; GP,
guanidinopropyl; GEGP, 3-[N-(2-guanidinoethyl)guanidine]-
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propyl; HUVECs, human umbilical vein endothelial cells; LCT,
liquid crystal templating; MSN, mesoporous silica nanoparticle;
PEI, poly(ethyleneimine); PEG, polyethylene glycol; RBCs, red
blood cells; RES, reticuloendothelial system; SWCNTs, single-
walled carbon nanotubes; TEM, transmission electron micro-
scope; WBCs, white blood cells
■
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