1166 Current Drug Targets, 2011, 12, 1166-1186

Multifunctional Mesoporous Silica Nanoparticles for Combined

Therapeutic, Diagnostic and Targeted Action in Cancer Treatment
Jessica M. Rosenholm1,2, Cecilia Sahlgren*,3 and Mika Lindén*,1

1
Center for Functional Materials, Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, FI-
20500, Turku, Finland
2
Nano Biomedical Research Center, Med-X Institute, Shanghai Jiao Tong University, 1954 Huashan Rd., 200030
Shanghai, P.R. China
3
Department of Biology, Åbo Akademi University, Artillerigatan 6A, FI-20520 Turku, Finland, and Turku Centre for
Biotechnology, University of Turku and Åbo Akademi University, P.O. Box 123, FI-20521, Turku, Finland

Abstract: The main objective in the development of nanomedicine is to obtain delivery platforms for targeted delivery of
drugs or imaging agents for improved therapeutic efficacy, reduced side effects and increased diagnostic sensitivity. A
(nano)material class that has been recognized for its controllable properties on many levels is ordered mesoporous
inorganic materials, typically in the form of amorphous silica (SiO 2). Characteristics for this class of materials include
mesoscopic order, tunable pore dimensions in the (macro)molecular size range, a high pore volume and surface area, the
possibility for selective surface functionality as well as morphology control. The robust but biodegradable ceramic matrix
moreover provides shelter for incorporated agents (drugs, proteins, imaging agents, photosensitizers) leaving the outer
particle surface free for further modification. The unique features make these materials particularly amenable to modular
design, whereby functional moieties and features may be interchanged or combined to produce multifunctional
nanodelivery systems combining targeting, diagnostic, and therapeutic actions. This review covers the latest developments
related to the use of mesoporous silica nanoparticles (MSNs) as nanocarriers in biomedical applications, with special
focus on cancer therapy and diagnostics.
Keywords: Mesoporous silica nanoparticles (MSNs), drug delivery systems, multimodal imaging, gene delivery, multidrug
resistance, photodynamic therapy, theragnostic agents, targeted cancer therapy.

1. INTRODUCTION monitoring (imaging) – often referred to as theragnostics. In

The application of nanotechnology in medicine is anti-
cipated to meet the therapeutic and diagnostic challenges that
today limit the therapeutic window and clinical applicability
of many drugs and hamper early diagnosis. A promising in
this field of research is the development of nanoparticle-
based systems for targeted both diagnostic and therapeutic
agents. Incorporation of drugs into the delivery platform
protects the drug from premature degradation and elimina-
tion and is important for clinical translation of poorly soluble
therapeutic agents, which are associated with numerous for-
mulation-related complications [1]. Novel drug delivery
technology is thus expected to improve bioavailability,
pharmacokinetics and tissue distribution of existing and
novel drugs.
Consequently, drug delivery is a central part of drug
development. New technologies are constantly being deve-
loped to meet the requirements of ‘smart’ drug delivery [2].
In cancer therapy, nanoparticles are expected to bring for-
ward multifunctional capabilities within the same system, i.e.
simultaneously function in targeted delivery of drugs to
cancer cells, in combination with concurrent real-time in vivo
*Address correspondence to this author at the Center for Functional
Materials, Department of Physical Chemistry, Åbo Akademi University,
Porthansgatan 3-5, FI-20500, Turku, Finland;
E-mails: cecilia.sahlgren@abo.fi, mika.linden@abo.fi
the beginning of the millennium, the research field of meso-
scopically ordered nanoporous silica-based materials entered
the drug delivery scene. The pore size and mesoscopic order
of these materials can be controlled using supramolecular
surfactant aggregates as structure-directing agents during the
synthesis, resulting in a highly porous, amorphous silica
material. The pore dimensions can be adjusted to fit the size
of payload molecules. A nanotechnology approach is used
for engineering of the Sol-gel derived, amorphous silica,
(SiO2), is known to be biocompatible and is suitable for
delivery of many types of biologically active agents,
including hydrophobic drugs, nucleic acids (DNA, siRNA)
and proteins, including hydrophobic drugs, nucleic acids
(DNA, siRNA) and proteins [3-15]. Due to their unique
properties, which include a narrow pore size distribution,
high specific surface area and pore volume along with
tunable pore sizes and mesoscopic order, mesoporous silica
particles have attracted attention after the first full reports of
the synthesis and characterization of this interesting class of
materials appeared in the early 90´s [16-18]. With the
advances in morphology control in the 2000’s, enabling
fairly straightforward synthesis of mesoporous silica nano-
particles (MSNs), not only the nanoscopic structure of
mesoporous silica but also the particles themselves could be
utilized as a nanoscale object. This development boosted the
interest for MSNs in biotechnological and biomedical
applications.

1389-4501/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

Multifunctional Mesoporous Silica Nanoparticles
Due to the large surface area and the controllable surface
functionality of MSNs, they can be loaded with large
amounts of drugs and coupled to molecules of choice for
targeting purposes, simultaneously carrying traceable (fluo-
rescent or magnetically active) modalities. Thus, MSNs are
truly multifunctional and can be used not only to specifically
deliver drugs (therapeutic action) but also for medical
imaging (diagnostic action). Targeted action that minimize
interactions with healthy tissue and enhances the cellular
uptake of the drugs in diseased cells, in combination with the
high drug load potential of MSNs will have a significant
impact on the therapeutic efficacy of drugs with limited
clinical applicability. The high drug load potential of MSNs
makes them efficient tools for multidrug delivery, a desirable
feature in cancer therapy given the complexity of oncogenic
activities. Furthermore, as the particles can be internalized
by cells, their use as carriers for contrast agents can sig-
nificantly prolong the imaging time-frame, as the half-lives
of many currently commercially available contrast agents are
extremely short and many are toxic. Thus, tissue specificity
is desired in order to improve the clinical diagnosis and also
because it is possible to reduce the dose, since the contrast
agent would become concentrated near the target. Conse-
quently, such drug delivery systems to target the delivery of
drugs or imaging agents to specific tissues and cells of the
body will not only have enormous impact on human health,
but will also be of great interest for medical imaging. In the
context of nanomedicine, many critical challenges related to
the development of medical technology for improved
diagnostic and therapeutic action are met by MSNs, which
will be outlined in the following.
2. MODULAR DESIGN OF MESOPOROUS SILICA
NANOPARTICLES
2.1. Particle Synthesis
The exponential growth in reports covering applications
of mesoporous silica in biological systems is due to recent
advances in morphology control of the materials, enabling
efficient particle size tailoring in the nanometer range [19].
Many mesoporous nanoparticulate fabrication routes are
dilute versions of the synthesis of MCM-41 (Mobil Com-
position of Matter No. 41), the by far most extensively
studied mesoporous material. The synthesis of MCM-41
relies on a cooperative self-assembly of supramolecular
aggregates of alkylammonium surfactants with polymerizing
silica species under alkaline (basic) conditions, eventually
leading to an organic-inorganic composite material consist-
ing of hexagonally ordered rod-shaped micelles surrounded
by an amorphous silica (SiO2) network. When the organic
portion is removed by either thermal treatment or solvent
extraction (ion exchange), an inorganic (ceramic) material
with hexagonally ordered voids (pores) with diameters of
approximately 3 nm is obtained. The pore size can be tuned
by for example changing the surfactant, the surfactant chain
length, or by adjusting the synthesis conditions. The reason
for why most nanoparticulate syntheses are based on this
particular synthesis is due to the fact that morphology
control is generally easier to achieve under basic conditions
than under acidic conditions. Spherical morphologies are,
however, readily achievable under acidic conditions albeit
Current Drug Targets, 2011, Vol. 12, No. 8 1167
usually on the micrometer scale, although MSNs has been
prepared successfully also under acidic [20], neutral [21],
and close to neutral conditions [22]. The addition of particle
growth quenchers is another useful adjunct for morphology
control on the nanoscale. Some crucial issues such as upscal-
ing, redispersibility after all production (functionalization)
steps as well as hydrolytic stability in physiological media,
remain to be properly addressed on the material design side
before MSNs are to be clinically applied, however the
diverse functionality and flexibility of MSNs as (potential)
drug carriers have been vastly demonstrated since the
beginning of the 2000’s.
2.2. Core-Shell Particles
In order to increase the functionality level of all-silica
MSNs, integration of mesoporous silica with nanocrystals of
other materials to form uniform core-shell composite nano-
structures with great potential in various biotechnological
and biomedical fields, especially medical imaging, have
emerged as a popular approach also outside the mesoporous
silica community. Mesoporous silica shells have thus been
successfully deposited on solid cores consisting of different
materials such as platinum [23, 24], gold [25-29], single
NaCl crystals [30], quantum dots [27, 31], and silver [32].
The mesoporous silica-coated composite material subject to
most extensive research is, however, beyond doubt magnetic,
typically ferro- or superparamagnetic iron oxide (SPIO) to
yield them magnetically active (for external control via an
applied magnetic field to achieve magnetically controlled
drug delivery, hyperthermia, or magnetic field-assisted
uptake of MSNs by cells) as well as suitable for magnetic
resonance imaging (MRI). The magnetic nanoparticles can
be incorporated/encapsulated into the mesoporous silica via
many fabrication strategies [33]. A frequently applied
synthesis route for production of materials with nanoscale
morphology is coating a silica shell around the magnetic iron
oxide nanocrystals to form a magnetic-core/silica-shell
structure. Hereby, the mesoporous layer can be deposited
either directly onto the magnetic cores [31, 34, 35], or then
the magnetic crystals can be pre-coated [36-39] with a dense
silica layer via the modified Stöber process [40, 41],
generally used to coat metal/metal oxide crystals with silica,
whereas the subsequent mesoporous coating process would
be equivalent to that of coating of silica surfaces [42-44].
The thus formed solid silica layer both protects the
underlying iron oxide from acidic environments (in which it
would dissolve) as well as facilitate the self-assembly of the
cationic surfactant templates on the negatively charged silica
surface [36]. However, we note that the synthesis of uniform
magnetic cores with outstanding magnetization and relaxi-
vity properties is probably the most challenging part in these
syntheses. Similarly as their all-siliceous counterparts,
obtaining still separable nanoparticles after all coating steps,
especially in the case of single crystal coating, can be a
challenge [34]. However, as the particles are magnetically
separable, sequential centrifugation and redispersion steps
are avoided, which affects the single particle integrity in
terms of redispersability in a favorable way. Two examples
of mesoporous silica coated magnetite (Fe3O4) particles of
different size are shown in Fig. (1).
1168 Current Drug Targets, 2011, Vol. 12, No. 8 Rosenholm et al.

a.

b.

Fig. (1). Examples of core-shell magnetite-mesoporous silica nanoparticles. a) A schematic representation of MR-imagable MSNs with a
SPIO nanoparticle core covered by a protective non-porous silica layer and a second layer of mesoporous silica. Drugs, fluorophores,
molecular MRI agents, and (responsive) coatings can be introduced by rational synthesis design. b) Transmission electron micrographs
(TEM) of SPIO nanoparticles-mesoporous silica particles of different types. Note that the overall particle size, the thickness of the
mesoporous silica layer, and the diameter of the SPIO nanoparticle core can be independently controlled. The micrographs were obtained by
a JEM 2010 (JEOL, Japan) instrument with 200 KV accelerated voltage. Synthesis and images Jixi Zhang, Nano Biomedical Research
Centre, Med-X Research Institute, Shanghai Jiao Tong University.

2.3. Flexible Surface Functionalization Strategies
The flexibility and modularity in the further design of
MSNs relies both on the mode of formation, structure, and
surface chemistry. Firstly, the modification of silica surfaces
is readily accomplished and fairly straightforward, including
methods such as surface grafting of functional silanes, poly-
mer coating, covalent linking of complexes, and co-
condensation. Mesoporous silica-specific functionalizations
have been also frequently reviewed in the literature [45].
Multifunctional Mesoporous Silica Nanoparticles
Secondly, the synthesis regime allows for in situ incur-
poration of functional groups into the silica matrix by
addition of functional silanes into the synthesis solution, thus
acting as part of the silica source. This mode-of-action result
in functional groups distributed fairly homogeneously
throughout the silica network, partly embedded in the pore
walls but also exposed on the pore wall surfaces, thus
accessible for guest molecules such as drugs. In synthesis
strategies where the functional silane acts also as a co-
structure directing agent [46] the fraction of functional
groups present on the pore walls can be enhanced.
Functional groups introduced via these co-condensation
strategies are frequently utilized to enhance interactions with
incorporated drug molecules (see below) or to couple
fluorochromes (or gadolinium-chelate complexes) to render
the MSNs imagable by optical (or MRI) methods. Thirdly,
the particle (outer) surface can subsequently be separately
functionalized in order to tune the particle surface charge (to
promote cellular uptake, reduce opsonization of plasma
proteins, direct the particles to specific intracellular
compartments, etc.), enhance particle suspension stability,
improve the ‘stealth’ properties of or introduce gate-keeping
properties to the nanoparticulate system, and to attach
targeting ligands to enable active targeting of the MSNs. In
order to protect the (inner) pore wall surfaces, reactive
functional groups can be attached to the particle surface
before surfactant removal in case the function in question
can withstand the conditions applied for extraction [47-49].
However, as the modifications used for further functionaliza-
tion of MSNs are often macromolecular such as polymers
(polyethylene glycol (PEG) for stealthiness [50], poly(D,L-
lactide-co-glycolide) (PLGA) as release retardant [51],
poly(allylamine) hydrochloride/poly(sodium-4-styrene sulfo-
nate) (PAH/PSS) multilayers for pH-responsiveness [52, 53],
poly(N-isopropylacrylamide) (PNIPAAm) for thermo-res-
ponsiveness [54, 55]) or proteins (antibodies or (poly)pep-
tides for targeting) the functionalization is mainly restricted
to the outer particle surface in any case, as the pore sizes for
MSNs are generally around 3 nm and thus cannot accom-
modate macromolecular substances. It has also suggested
that the most kinetically available Si-OH groups are those on
the outside surfaces of the silica particles, and hence also
kinetically more accessible for functionalization [56] and
thus preferential modification of the particle surface can be
achieved also after extraction of the surfactant template. To
keep the beneficiaries of a MSN system, a key issue is that
the pore network is still available after these functionaliza-
tions, as the loading of cargo (drugs) is generally performed
after these steps.
2.4. Attachment of Targeting Moieties
The attachment of cell-specific targeting ligands to the
outer surface of the cells is a common method for enhancing
MSN cell-specificity. If the targeting moiety is a small-
molecular ligand, such as sugars or folic acid, the attachment
of the ligand can be performed before drug loading and in
either organic or aqueous solvent, of which organic solvent
might be preferential to not alter the silica matrix. In the case
of proteinous targeting moieties (such as polypeptides or
antibodies), on the other hand, the conjugation step is most
likely to be performed last and moreover in aqueous solvent,
to preserve the activity of the protein, and thus the drug
Current Drug Targets, 2011, Vol. 12, No. 8 1169
should not leak out during this conjugation step. However, as
the MSN should at this point anyhow be designed not to
release its cargo immediately upon immersion in aqueous
media (i.e. when exposed to physiological conditions), it is
in line with rational processing of the MSNs for drug
delivery. As standard bioconjugation reactions such as 1-
ethyl-3-(3-dimethylaminopropyl)-carbodiimide- (EDC) or
N,N'-Dicyclohexylcarbodiimide- (DCC) based coupling (or
other peptide synthesis reactants) are mostly utilized for the
attachment, reactive groups should be readily accessible on
the particle surface. Most often amino groups are utilized for
this kind of conjugation, but also -OH, -COOH, or -SH
groups may be utilized. Fluorochromes are attached to the
particle system using similar chemistry, generally via amine-
specific reactions, but in the case of small-molecular ligands
the fluorophores may benefit from being embedded “deeper”
into the particle structure, in order not to have several bulky
molecules onto the particle surface which may hamper or
affect the binding (targeting) ability of the biospecific
molecule. In the case of a “middle layer”, either a responsive
polymer layer or PEG terminal functional groups of the
polymer chains may thus be essential. In many cases the
targeting ligand can be attached to the polymer chains before
conjugated to the particles, whereas bifunctional polymers
would be advantageous in order to attach the ligand to one
end of the polymer chain and “reserve” the other end for
particle conjugation (or use protective groups) to attain
chemoselectivity. PEG is readily commercially available
with different (bi)functionalities for this purpose. When the
ligand is attached as the last step, depending on the func-
tional groups of the ligand, bridging between particles (self-
conjugation) should be avoided. This is also crucial if a poly-
mer chain is conjugated to the particles with monofunctional
terminal ends (the same functional group in both ends of the
chain) or in the use of bifunctional linker molecules.
Importantly, as the ligand-receptor binding is conformation-
defined, it is crucial the ligand is attached to the particle in a
way that it retains its binding activity. Thus, for proteinous
ligands, also the conditions applied in the conjugation step
must prevent denaturation of the protein. As bioconjugation
protocols are normally adapted for biomolecules, this
procedure is quite straightforward and the challenge rather
lies in playing with the chemistries in a modular fashion to
have a reproducible functionalization protocol sequence
compatible with the drug loading process.
3. MSNS AS DRUG DELIVERY VEHICLES
3.1. Loading of Biologically Active Cargo into Mesopo-
rous Silica
One of the main strengths of MSNs in delivery applica-
tions is the possibility to carry large amounts of payload due
to their high intrinsic specific surface areas and pore
volumes. Drug loading degrees exceeding 30 wt% have been
reported for mesoporous silica, and keeping in mind that the
density of amorphous silica is about 2.2 g/cm3, this value
would correspond to loading degrees exceeding 50 wt% for
polymeric particles assuming a polymer density of 1 g/cm3.
Furthermore, the drug loading levels can be rationally
controlled, which means that both the drug loading as well as
the particle concentration can be used as parameters to
control the total drug levels in vitro and in vivo. This is
1170 Current Drug Targets, 2011, Vol. 12, No. 8
important, as the cell selectivity in the targeting process as
well as the particle toxicity are particle concentration-
dependent.
As mentioned above, the loading of drugs into and/or
onto MSNs is typically carried out as a separate step
following particle synthesis. Separation of material synthesis
and drug incorporation allows independent optimization of
structural and physicochemical properties of the carrier and
the conditions used for the drug loading. Incorporation of
cargo into silica can be achieved by different means; the
three most important being physical adsorption from solution
into the mesopores, physical adsorption from solution onto
the outer surface of the MSNs, and covalent linking. Fur-
thermore, also impregnation by incipient wetness, as well as
loading from a drug melt has been studied. The molecular
structure as well as dimensions of the cargo with respect to
the diameter of the mesopores, its water solubility (and
cytotoxicity), and the chemical stability of the cargo deter-
mine which of these methods is the preferred one. In the
following we will briefly discuss the different means of
cargo incorporation into mesoporous silicas. The discussion
is based on selected examples highlighting the mechanistic
aspects related to each method. As most of the published
mechanistic studies have been performed on microparticles
rather than on MSNs, much of the discussion is based on
microparticles. However, the mechanistic aspects are largely
valid for both particle size ranges, and results obtained for
MSNs are included whenever considered relevant. The same
argumentation holds for the cargo, as studies directly aimed
at cancer therapy represent only a fraction of the published
papers related to controlled adsorption-desorption (loading-
release) of bioactive cargo from mesoporous silica.
3.1.1. Adsorption from Aqueous Solution: Small-Molecular
Drugs
The most often used method for the loading of small
drugs into the pores of MSNs is physical adsorption from
solution. The surface silanol groups, ≡Si-OH, always present
on the silica surface, and which typically serve as adsorption
sites, are present at surface concentrations of at least 2-4
groups/nm2 [57]. Therefore, the surface concentration of
adsorption sites is normally not a limiting factor for the
extent of adsorption. The silanol groups are amphoteric, and
can be present in both a protonated, ≡Si-OH2+, and a
deprotonated form, ≡Si-O-, in aqueous media depending on
the pH [58]. The point of zero charge for silica, i.e. the pH at
which the net-surface charge is zero, is about 2-3, which
means that in the absence of specific ion adsorption, the
silica surface is negatively charged under biologically
relevant conditions. Therefore, electrostatic adsorption of
positively charged adsorbates (i.e. in this case, drugs) is an
attractive method for incorporating water-soluble cargo into
siliceous MSNs. The extent of electrostatic adsorption can be
further increased by introducing functional groups, very
often weak acids and bases like carboxylic acids or amines,
onto the silica surface, which can be used to tune the
effective surface charge under given pH conditions, and also
provide the possibility for additional specific adsorbate-
adsorbent interactions. The extent to which the effective
surface charge at a given pH is altered is naturally a function
of both the chemical nature of the introduced functions and
their surface concentration. This fact can be utilized for
Rosenholm et al.
efficient pH-dependent adsorption, as was recently shown
for the adsorption of the highly water soluble anti-
inflammatory drug salicylic acid [59]. High amounts of
salicylic acid could be adsorbed from aqueous solvent onto
amino-functionalized mesoporous silica at pH = 3, up to 16
µmol/m2 when the silica surface was functionalized by
surface hyperbranching polymerization of polyethyleneimine
(PEI), while negligible amounts salicylic acid was adsorbed
on native silica under the same conditions. The high surface
concentration of amino groups lead to strongly positive net-
surface charges even under low pH-conditions, and also
provide the possibility for specific amine-carboxylic acid
interactions. Under neutral conditions the extent of adsorp-
tion was much lower due to the presence of negatively
charged silanol groups, suggesting that the pH-swing
methodology could be an attractive means for drug adsorp-
tion from aqueous solution. The adsorption of famotidine, a
histamine H2-receptor antagonist that inhibits stomach acid
production, into mesoporous silica from 50/50 v/v methanol/
water solutions at pH = 7 was studied by Tang et al. [60].
Famotidine contains four amino-groups in its molecular
structure, and the adsorbed amount could be directly
correlated with the amount of carboxylic acid groups present
on the functionalized mesoporous silica. No adsorption of
famotidine was observed for corresponding native silica
materials, although the drug and the silica should be
oppositely charged under the studied conditions. These
results further stress the importance of adequate surface
modifications to optimize the adsorbate-adsorbent interac-
tions. The adsorption process could be satisfactorily des-
cribed as Langmuir adsorption under the used constant pH-
conditions, suggesting monolayer adsorption to equivalent
adsorption sites, and that there was little interaction between
the adsorbed molecules. The monolayer capacity of the
mesoporous silica with the highest carboxylic acid group
content was 340 mg famotidine/g silica. The fact that the
adsorption can be satisfactorily modeled also means that the
drug loading degree can be rationally fine-tuned to be any
loading degree between zero and the maximum loading.
3.1.2. Adsorption from Aqueous Solution: Macromolecular
Species
The loading of biomacromolecules is, naturally, normally
carried out from aqueous solution in order to maintain the
biological activity. Many studies have been published related
to the adsorption of proteins and enzymes into the pores of
mesoporous silica. Most of them are aimed at permanent
immobilization of enzymes to create bionanoreactors, and
thus fall outside the scope of the present review. The
interested reader is referred to two recent reviews dedicated
to this topic [61, 62]. In the following, we will briefly cover
some mechanistic aspects of the incorporation of biomacro-
molecular species into and onto mesoporous silica.
3.1.3. Adsorption of Proteins
Balkus and co-workers were the first to publish a work
on the immobilization of globular proteins (cytochrome C,
papain, and trypsin) on ordered mesoporous silica with pore
sizes in the range of 3 nm [63]. Deere et al., on the other
hand, were the first to report adsorption isotherms at 25 °C
of cytochrome C on different mesoporous silica molecular
sieves [64, 65]. The isotherms were recorded at a pH of 6.5
Multifunctional Mesoporous Silica Nanoparticles
and are of the Langmuir type. Release of cytochrome C by
washing could be achieved only if the pH was raised to 10,
which is close to its pI, i.e. the pH value in solution at which
the sum of the charges on the protein is zero. A maximum
loading of 10.2 µmol/g was achieved under the conditions
employed in this study. Hartmann et al. showed that the
extent of adsorption of both cytochrome C [66] and lyso-
zyme [67] onto mesoporous silica (the studied mesoporous
silica supports had pore diameters in the range 3-11 nm) is
strongly pH dependent, and a maximum loading degrees
were shown to occur close to the pI of each protein. This
observation was attributed to a minimum of electrostatic
repulsion between adsorbed proteins, favoring protein close
packing inside the mesopores. Furthermore, under these
“charge-neutralized” conditions, hydrophobic adsorbent-
adsorbate interactions increase in importance, and the protein
is also compact due to lacking internal charge repulsion
effects. Hydrogen bonding and hydrophobic effects can be
also utilized for efficient adsorption of macromolecular
active agents into mesoporous silicas, as recently shown for
the incorporation of lipase into mesoporous silica func-
tionalized by alkyl-groups [68]. The lysozyme and cyto-
chrome C adsorption data could be generally fitted using a
Langmuir adsorption isotherm [66, 67]. Introduction of Al
into the silica network lead to increased levels of adsorption,
and this was ascribed to the presence of additional negative
charges at the Al-sites. The maximum loadings were about
680 mg lysozyme/g silica and about 500 mg cytochrome
C/silica g under the studied conditions. It was concluded that
the proteins were mainly located into the mesopores in all
cases, but that differences in the pore volume of the supports
accounted for the observed differences in adsorption capa-
city. Stucky et al. [69] reported the adsorption and desorp-
tion at pH = 7 of conalbumin (MW 77 kD, pI 6.0), chicken
egg ovalbumin (MW 44 kD, pI 4.9), and soybean trypsin
inhibitor protein (MW 14 kD, pI 5.2) onto/from amino-func-
tionalized mesoporous silicas with different pore diameters
(5.9 nm, and 16 nm). While the smaller pore material mainly
adsorbed the smallest protein, the trypsin inhibitor, the three
proteins studied were adsorbed by the larger pore material.
The proteins were tightly associated with the support due to
attractive electrostatic interactions, but could be released by
increasing the ionic strength of the solution. All the studies
discussed above have been conducted using microparticles
of mesoporous silica. However, high loading degrees of
cytochrome C has been also reported for MSNs when the
adsorption was performed from aqueous solution into MSNs
having pore diameters around 5 nm [70]. A maximum
loading of 415 mg cytochrome C/g silica could be achieved,
but at the expense of structural integrity of the MSNs. A
loading degree of 95 mg cytochrome C/g silica could be
reached without loss of structural order. No reason for the
loss of order at high loading degrees was given, but we
tentatively ascribe it to concurrent hydrolytic degradation of
the MSNs due to silica dissolution, which would be more
rapid/pronounced from MSNs than for micron-sized par-
ticles.
3.1.4. Adsorption of Genes
The adsorption of genes into mesoporous silicas has also
been studied. For example, linear DNA with a green fluore-
scent protein (GFP) sequence with approximately 760 base-
Current Drug Targets, 2011, Vol. 12, No. 8 1171
pairs has been successfully incorporated into the mesopores
of mesoporous silica by adsorption from aqueous solution
[71]. It was shown that pre-incorporation of divalent metal
ions into the pores enhanced the adsorption of DNA, as the
metal ions served as charge mediators between the nega-
tively charged silica surface and the negatively charged
DNA. The adsorption of DNA could be further enhanced by
functionalization of the pore walls with amino groups, and
DNA loading degrees of 15.7 mg/g silica could be reached
for mesoporous silica materials with pore dimensions of 10
nm. For native silica materials with mesopore dimensions of
about 5 and 10 nm the adsorption process could be described
as Langmuir adsorption, while the adsorption process was
better described by the Freundlich isotherm for silicas with 3
nm mesopores. This suggests that the intermolecular
interactions cannot be neglected in the latter cases.
However, in many cases the pore diameters of MSNs are
in the range of 2-3 nm, which severely limits the possibility
for adsorption of macromolecules into the mesopores. There-
fore, adsorption of genes or proteins onto the outer surface of
MSNs has been also studied. The underlying driving forces
for adsorption to the outer surface of the particles are largely
the same as for adsorption into mesopores, although the
influences of confinement effects are naturally absent. Nel et
al. [72] adsorbed siRNA and DNA constructs to MSNs that
has been firstly coated with a layer of PEI, a well-known
gene transfection agent. Particle/nucleic acid ratios of 10-100
were needed for complete adsorption. As expected, the high
positive charge density of the PEI layer was beneficial for
adsorption, and corresponding particles coated with an
organophosphate layer exhibited a much lower nucleic acid
adsorption capacity.
3.1.5. Adsorption from Non-Aqueous Solution – Poorly
Water Soluble Small-Molecular Drugs
Many attractive drug candidates have low water solu-
bility, and therefore, in these cases loading of the active
component by adsorption from aqueous solution is typically
not an option. Furthermore, as MSNs are inherently
biodegradable, silica dissolution during the loading process
under aqueous conditions could lead to unwanted structural
changes of the carrier. Therefore, drug incorporation by
adsorption from non-aqueous solvents has been widely
applied. In these cases, electrostatic interactions are typically
less important, and the driving forces for adsorption are
normally hydrogen-bonding or polar interactions. Also in
this case, the strength of these interactions can be enhanced
by introducing surface groups onto the mesoporous support
which have a strong affinity for the adsorbate (drug), like
amino groups in the case of adsorption of carboxylic acids.
The choice of solvent is mainly determined by the solubility
and stability of the biologically active component. However,
the solvent properties also have a great influence on the
adsorption process itself, as the solvent molecules compete
for adsorption sites on the silica surface. Water molecules
naturally also compete for the adsorption sites under aqueous
conditions, but in those cases the electrostatic adsorbate-
adsorbent interactions are typically dominant. In addition,
solvent-adsorbate interactions could play an important role
for the extent of adsorption, as the driving force for adsorp-
tion is lower in good solvents. For example, more than 30
wt% of the anti-inflammatory agent ibuprofen can be incur-
1172 Current Drug Targets, 2011, Vol. 12, No. 8
porated into mesoporous silica by adsorption from hexane if
the pore size is not limiting the extent of adsorption [73, 74]
while the corresponding value is 7 wt % if the adsorption is
carried out from methanol [75]. Methanol is a very good
solvent for ibuprofen, and is also capable of forming
hydrogen bonds with the surface silanols. Furthermore, the
maximum amount of ibuprofen adsorbed from the aromatic
solvent toluene to bare mesoporous silica is about 0.2
µmol/m2, whereas the corresponding value from the aliphatic
solvent hexane is 1.6 µmol/m2 [59]. In aromatic solvents,
attractive π-π interactions between the solvent and aromatic
adsorbents can occur, decreasing the tendency towards
adsorption. Probably even more importantly, there are
attractive interactions between the π electrons in the aromatic
ring of toluene and the protons in the carboxylic acid group
as well as in surface silanols, decreasing the tendency
towards adsorption even further. The solvent-adsorbate
interactions are much weaker and dominated by dispersion
forces in many aliphatic solvents, like n-hexane and cyclo-
hexane, and the lower achievable loading degree of ibupro-
fen from toluene as compared to hexane can be therefore
understood. It has also been shown that for the adsorption of
ibuprofen from hexane, for supports of a given pore size, the
surface area rather than the pore volume is determining the
maximum loading capacity [73].
Furthermore, a marked influence of the solvent polarity
on the extent of drug adsorption to mesoporous silica has
been observed. Kuroda and co-workers studied the adsorp-
tion of the anticancer agent taxol into mesoporous silica
from different solvents, and found that this chemotherapy
agent was adsorbed from dichloromethane or toluene, but
not from methanol or acetone [76]. This finding was related
to the different polarities of the solvents, and especially to
the proton-acceptor solubility parameter, which is clearly
lower for toluene and dichloromethane than for methanol
and acetone. Taxol contains both hydroxyl groups and carbo-
nyl groups that are possible acceptors for hydrogen bonding.
Therefore, it has a good affinity with solvents having a high
proton acceptor capacity, and attractive solvent-adsorbate
interactions, together with preferential solvent-adsorbent
interactions through hydrogen bond formation with surface
silanols, decrease the extent of taxol adsorption.
In most studies, the adsorption of poorly water-soluble
drugs into mesoporous silica has been carried out using a
fixed high concentration of the drug in solution, as a high
drug loading degree has been aimed for. However, some
detailed adsorption studies have been also reported. The
adsorption isotherms for ibuprofen adsorption to mesoporous
silicas from n-hexane have been determined. Here, it is
shown that the adsorption can satisfactorily be described
using the Langmuir adsorption isotherm, suggesting a
monolayer adsorption [74]; which is why the adsorption
process is dependent on the surface area rather than the total
pore volume, for situations where the pore diameter is not
limiting the extent of adsorption.
The main drawback associated with impregnation from
organic solvents is the possibility for remaining solvent
residues after solvent evaporation [77]. Thus, the use of
other fluids, like supercritical [78] or near-critical [79] CO2,
as the solvent for the biologically active component is a
promising alternative to organic solvents in the drug loading
Rosenholm et al.
process. However, we note that in many studies related to
MSNs, amino-functionalized MSNs have been used. CO 2
interacts specifically with amino-groups, why any possible
alterations of the surface chemistry of amino-functionalized
MSNs due to CO2 chemisorption to amino-groups should be
taken in to the account when using liquid CO2 as the solvent
for drug loading into amino-functionalized MSNs.
3.1.6. Covalent Linking of Cargo to the Support
Covalent linking of the cargo to the mesoporous silica
carrier is an attractive alternative to physical adsorption,
especially when the cargo has non-negligible water solubility
and/or the cargo is highly cytotoxic, in order to prevent
premature leaching of drug. Furthermore, the loading
degrees observed for MSNs are often lower than those
obtained for corresponding microparticles. This may be
related to the fact that large amounts of physically adsorbed
cargo may be leached out during the washing step, as for 100
nm particles about 50 % of the pore volume is contained in
the outermost 10 nm of the particle [80]. There are, however,
not so many studies published to date where covalent linking
of the cargo to mesoporous silica has been performed with
the aim of being able to subsequently release the cargo. This
may be connected to the fact that it in most cases is essential
that the biologically active molecules are reformed into their
original chemical structure upon detachment. Furthermore,
there has to be a mechanism by which the cargo is released
when the carrier particle has reached the target site. The
covalent linking of ibuprofen to amino-functionalized meso-
porous silica has been demonstrated, but no release data was
shown [81]. In a recent interesting study by Lin and co-
workers [82], a cysteine residue was covalently attached to
thiol-functionalized MSNs, which were effectively interna-
lized by HeLa cells. Covalent attachment of the cysteine by
S-S-linkage to the silica nanoparticle prevented premature
release in the extracellular media, but cysteine was released
inside the cells due to reduction of the S-S bond. Due to high
intracellular concentrations of gluthatione, the intracellular
redox-potential is much higher compared to the outside
mileu, thereby providing an inbuilt differential release
mechanism. Another example for successful covalent linking
of the biologically active agent where subsequent release has
been demonstrated is the covalent linking of the chemother-
apeutic agent methotrexate (MTX) to amine-functionalized
MSNs [83]. It has previously been shown that MTX does not
adsorb to silica, and that alumina has to be introduced into
the silica framework to enhance the drug adsorption degree
[84]. However, covalent linking of MTX to silica was suc-
cessfully accomplished using standard EDC coupling linking
a carboxylic acid group present in the MTX molecular struc-
ture to amino groups present on the mesoporous silica. The
amino-groups were incorporated either by co-condensation
or by surface hyperbranching-polymerization of PEI. Pre-
mature (extracellular) release could be prevented, and the
intracellular detachment of MTX from the support in vitro
was mainly ascribed to enzymatic degradation of the amide
bond in the lysosomes.
3.1.7. Additional Methods for Loading of Cargo
Incipient wetness has also been used for the loading of
drugs into mesoporous silica [85]. Here, the drug is dis-
solved into a suitable solvent and just as much liquid volume
Multifunctional Mesoporous Silica Nanoparticles
that can be accommodated in the available pore volume of
the mesoporous silica support is used in the loading process.
Both itraconazole and ibuprofen were loaded into meso-
porous silica with pore dimensions of 8.4 nm using this
technique. In comparison with physical adsorption from
dichloromethane, the incipient wetness impregnation method
favored the positioning of itraconazole molecules inside
intra-wall micropores, while the solvent method favored
their positioning on mesopore walls. Furthermore, the same
group also studied the loading of both model drugs from a
melt of the drug [85]. While the high viscosity of the
itraconazole melt prevented loading, ibuprofen was
successfully incorporated at lower loading degrees using this
method, and was shown to be preferentially located in intra-
wall micropores. At higher loading levels, formation of
larger particles of ibuprofen located outside the silica
mesopore system was observed.
3.1.8. Concurrent Loading of Several Active Cargos
It is also possible to combine the means described above
for the loading of two or more biologically active com-
ponents into MSNs. This is an extremely important feature,
as efficient next-generation cancer therapies based on che-
motherapy in combination with controlled release systems
will most likely involve the co-delivery of two or more
active agents. The most straightforward means is, if possible,
to adsorb several active components in parallel, as recently
demonstrated for the loading of two different hydrophobic
model drugs from cyclohexane [86]. In another recent study,
the antitumor drug doxorobucin (DOX) was first adsorbed
onto cyanato-functionalized MSNs from a methanol/water
mixed solvent, after which a second generation polyamido-
amine dendrimer was covalently attached to the outer surface
of the MSN particles by urea linkage to cap the pore open-
ings [87]. A DOX loading as high as 40 wt % was obtained.
siRNA was then adsorbed to the dendrimer-functionalized
outer surface of the DOX-loaded MSN particles. In another
recent study, Lin and co-workers first synthesized boronic
acid-functionalized MSNs, after which cyclic adenosine
monophosphate (AMP) was adsorbed into the mesopores
from phosphate buffered saline (PBS) [88]. Gluconic acid-
modified insulin was subsequently covalently linked to the
boronic acid groups present on the outer surface of the
MSNs. The loadings of cyclic AMP and gluconic acid-
functionalized insulin were reported to be 27 and 64 µmol/g,
respectively.
3.2. Cargo Release from Mesoporous Silica
3.2.1. Non-functionalized Mesoporous Silica: Simple
(Matrix) Diffusion
In most cases, the release of small-molecular cargo incur-
porated into the pores of mesoporous silica is diffusion-
controlled and follows first order kinetics in the absence of
strong adsorbate-adsorbent interactions. Important parame-
ters influencing the release rate are the effective pore size,
the polarity of the pore system, the water solubility of the
cargo, and state of the cargo in the pores (amorphous or
crystalline) which also has an influence on the cargo
dissolution kinetics. Furthermore, the particle size will have
an important influence on the release rate, and faster overall
release rates can be expected from MSNs than for corres-
Current Drug Targets, 2011, Vol. 12, No. 8 1173
ponding microparticles. However, the release rate and also
the release mechanism are strongly influenced by the mag-
nitude of attractive cargo-silica interactions. Bergström et al.
[89] studied the release of cationic and anionic dyes from
mesoporous silica spheres into PBS by confocal microscopy
on single particles, an approach originally developed by the
group of Landry for the characterization of differential
functionalization of mesoporous silica [90]. For the anionic
dye, which does not interact strongly with the silica, the
release was controlled by simple diffusion. The release of the
cationic dye, on the other hand, followed a distinctively
different pattern, as a significant fraction of the remained dye
permanently adsorbed onto the pore walls of the negatively
charged mesoporous silica spheres, predominantly near the
surface of the spheres. This formation of a semipermeable
“skin” close to the particle surface led to delayed release of
the dye molecules, and consequently the release event could
not be described by standard diffusion models. Although the
results were obtained for spherical mesoporous particles
several micrometers in diameter and the effects may be
smaller for MSNs, these interesting results highlight the
interdependency between the adsorbate-adsorbent interact-
tions and the release profiles. Furthermore, the pore size and
number of available adsorption sites with respect to the
molecular size of the cargo has been recently shown to
define the relative influence of attractive adsorbent-adsorbate
interactions, and also on the subsequent release kinetics.
Bräuchle et al. [91] studied the mobility of the DOX inside
dried, mesoscopically ordered silica-surfactant films, and
found that this anticancer drug was immobile in the
cylindrical channels of films templated by the non-ionic
surfactant brij® 56, while it was highly mobile inside corres-
ponding channels templated by the relatively bulky non-
ionic surfactant pluronic® P-123. Furthermore, mobility was
also observed inside films templated by the smaller cationic
surfactant cetyltrimethylammonium bromide (CTAB) which
has a molecular dimension comparable to that of brij® 56.
These findings were attributed to the blocking of silanol sur-
face groups by the cationic surfactant, hindering hydrogen-
bonding between oxygen atoms present in the molecular
structure of DOX and the silica surface, while free silanols
were present in the films templated by the non-ionic
surfactants. The difference between the DOX mobility inside
the pluronic® P-123 and Brij® 56 templated films, on the
other hand, was attributed to a reduced influence of
hydrogen bonding in larger channels. The level of DOX
mobility was directly reflected in the rate of diffusion-
controlled DOX release from the films.
However, in many cases a slower drug release than
expected for first-order release kinetics is observed at longer
times, even in the absence of strong attractive adsorbate-
silica interactions. This has been attributed to differences in
cargo-support interactions between the inner and outer
portion of the surface. Other suggested reasons for this
observation has been the deposition of hydroxyapatite
crystals onto the silica surface leading to partial pore
blocking, when ibuprofen release from mesoporous silica
was studied in a simulated body fluid [92] as well as for
empty MSNs [93], in agreement with previous considera-
tions. However, recently an alternative explanation was
brought forward to account for the clearly slower ibuprofen
release from MSNs at longer times. It is based on silica
1174 Current Drug Targets, 2011, Vol. 12, No. 8
Fig. (2). Release of ibuprofen from pristine (non-functionalized) mesoporous silica (left) in simulated body fluid (SBF) along with its square-
root of time plot (right), indicating a diffusion-controlled release process. (From: Rosenholm JM. Modular design of mesoporous silica
materials: towards multifunctional drug delivery systems. DSc(Tech) thesis. Turku: Abo Akademi University 2008).
dissolution and preferential re-deposition close to the pore
entrances leading to partial pore blocking [94]. Indeed, silica
does dissolve under physiological conditions, and this is a
much wanted property of the material in drug delivery
applications using MSNs. However, this is often overlooked
in release studies and typically the silica dissolution kinetics
is not reported. The dissolution kinetics may be different for
silicas prepared under different experimental conditions, and
is especially dependent on if the silica has been thermally
treated or not, as the degree of silica condensation is en-
hanced by thermal treatment. The dissolution-reprecipitation
mechanism is supported by the fact that a marked decrease in
pore volume was observed, although the mesostructure
appeared intact as determined by X-ray diffraction (XRD)
for particles with a fairly broad size-distribution of 200-800
nm. One may expect that this is another parameter that leads
to faster kinetics from smaller particles than from larger
particles with otherwise similar structural and surface
chemical properties.
The molecular-level interactions influencing the release
of macromolecular cargo from mesoporous silica is naturally
more complex due to the presence of a multitude of potential
binding sites and also due to the possibility for molecular
weight- or loading degree-dependent conformational and
polarity differences. Very few mechanistic studies related to
the release of macromolecular cargo from mesoporous silicas
have been reported. However, in a recent study, the release
of fluorescently labeled dextrans, a commonly used model
for proteins, from microparticles of mesoporous silica with a
pore size of 6.5 nm was described [95]. Two dextrans with
different molecular weights, 3 and 10 kD, were used. As
expected, the release rate of the high-molecular weight
dextran was slower than that of the low-molecular weight
dextran, but the release curves had a similar shape
suggesting that the release mechanism was similar in both
cases. It was concluded that dextran release was not dif-
fusion-limited. The release followed a logarithmic time
dependency normally observed for chemisorption events,
and the dextran release process was discussed in terms of
parallel adsorption and desorption events coupled to concen-
Rosenholm et al.
tration-dependent effective diffusion constants. In another
recent study, the release of the antibiotic vancomycin from
mesoporous silicas with different pore diameters was studied
[96]. For the pore size range studied, 4-9 nm, variations in
the pore diameter had little effect on the drug release rate.
However, the pore morphology and the effective length of
the pores, which naturally is directly related to the diffu-
sional path length, had clear influences on vancomycin
release rate, as expected for a diffusion-controlled release.
Again, these results highlight that the release rate of macro-
molecules from MSNs may be faster than that of corres-
ponding microparticles. This observation is also perfectly in
line with diffusion theory, which furthermore is charac-
terized by a fast release in the beginning with gradually
(square-root of time dependent) decreasing rate of diffusion
(release), see Fig. (2). As diffusion-controlled systems are
furthermore often accompanied by an initial “burst” release,
this has raised the need for means to minimize early release
of the active component from MSNs, and in the best case
completely hinder a pre-mature release before the cargo has
reached its site of action. This will be discussed in the next
section.
3.2.2. Stimuli-Responsive Release Systems Based on MSNs
The value of having the most selective targeting system
is of course lost if the cargo is released from the carrier
before it reaches the target site. For chemotherapy applica-
tions it is desirable that no release of cargo should occur in
the extracellular environment, while a fast release of the
cargo should occur once the carrier particle has reached its
point of action. As discussed above, the release of cargo
from mesoporous silica is often diffusion-controlled and
typically associated with a pronounced initial burst. There-
fore, there is a strong current interest in the development of
means to prevent a fast initial release of the cargo, which in
the optimal case would be sensitive to the local environment
of the carrier particles in a way that release of the cargo is
only triggered inside the cells or within specific cellular
compartments after internalization. In addition to the
reversible covalent linking of drug to the mesopore surface
already discussed above, many other inherent or stimuli-
Multifunctional Mesoporous Silica Nanoparticles
responsive systems have been reported. The development in
this field up to 2009 was recently reviewed [97, 98], and in
the following we will therefore only give some selected
examples of stimuli-responsive systems which could operate
under biologically relevant conditions. These can be divided
into two main classes; a) systems which can be triggered by
external stimuli, such as ultraviolet light, and b) systems
which should react to the differences in chemical conditions
between the extra-cellular and intra-cellular environments,
such as differences in pH, redox potential, or enzymatic
activity. We will mainly focus on systems related to the
latter class in the following.
3.2.2.1. pH-Responsive Systems
The means of MSN administration determines which
type of functionality is needed. For example, for pH-res-
ponsive systems, the desired pH-response is in most cases
opposite for oral and intravenous administration. In order to
prevent premature release in the gastric tract, the mesopores
should remain closed at under acidic conditions and open
under more pH neutral conditions, for instance by some gate-
keeping moieties. Such a pH-response has been demons-
trated for MSNs onto which polyamines in the form of
silanes were covalently linked to act as gate-keepers [99].
The mesopores remained closed under acidic conditions due
to the “swollen” conformation of the polyamines when
charged, thus hindering the release of the cargo; but release
of vitamin B was observed under pH neutral conditions to an
extent which was dependent on the ionic composition of the
medium [100]. Both pH-induced changes in the extent of
hydrogen-bonding interaction between amines (neutral pH,
open gate) and Coulombic repulsions (acidic pH, closed
gate) and the formation of anion-amine complexes were
suggested to account for this observation. Also electrostatic
assembly of poly(acrylic acid) to amino-functionalized me-
soporous silica has been shown to effectively entrap proteins
under acidic conditions, while about 35 % of the encap-
sulated protein was released at pH 7.4 within 10 h [101].
Another example of a polymer-based coating showing a
corresponding pH-response is the pH-dependent release of
ibuprofen from mesoporous silica spheres coated with a co-
polymer layer consisting of poly(methacrylic acid-co-vinyl
triethoxylsilane) [102]. Here, the silane function links the
polymer covalently to the silica, while the pH-dependent
protonation of the carboxylic acid groups in the metharylate
control the degree of polymer swelling. Recently, a MSN
system with charge-convertible, pH-hydrolyzable groups on
the pore walls was also reported [103]. The employed
citraconic amide functionality possesses a negative charge at
pH 7.4 (extracellular conditions) but undergoes charge con-
version at acidic pH 5 (endosomal conditions). The feasi-
bility of this mechanism was demonstrated by release of
cytochrome C, which was more pronounced at pH 5 due to
the proteins effective positive charge at this pH.
On the other hand, for intravenous administration, which
is likely to be the most frequently used administration route
for MSN-based chemotherapy the pores should preferentially
remain closed under the neutral pH conditions prevailing in
the blood stream, while the pores should be open to the
outside under the more acidic pH conditions prevailing in
tumor sites and intracellular compartments like endosomes
and lysosomes. One early example of such a pH-responsive
Current Drug Targets, 2011, Vol. 12, No. 8 1175
system is the adsorption of poly(dimethyldiallylammonium
chloride) to carboxylic acid-modified mesoporous silica
[104]. Vancomycin was used as the model drug, and
pronounced release of entrapped vancomycin was observed
at pHs ≤ 4.5, while little drug release was observed at pH
6.5. On a related note, chitosan has been also shown to pro-
vide pH-sensitive molecular gate properties, as demonstrated
by the release of insulin from mesoporous silica/silicon films
[105]. Zero-release was observed under pH-neutral con-
ditions, while insulin was released at pH 6 due to swelling of
the chitosan hydrogel. Furthermore, a pH-dependency was
recently demonstrated using a molecular machine-concept
based on [2] pseudorotaxane which was covalently linked to
MSNs. A model fluorescent dye remained inside the
mesopores under pH-neutral conditions, while a release of
cargo was observed upon decreasing the pH below 5, which
is close to the pH in lysosomes [106]. Another example of
corresponding pH-responsive systems is gold nanoparticle-
capped mesoporous silica, where the gold nanoparticles were
covalently linked to the outer surface of the mesoporous
silica through an acid-labile acetal linker [107]. Zero-release
of a dye used as the model cargo was observed under neutral
conditions, while the dye diffused out of the pores upon
lowering of the pH. Almost complete release of the cargo
was observed within 10 hours at a pH of 4.0.
3.2.2.2. Redox-Sensitive Systems
Another often used means for stimuli-responsive release
of cargo from MSNs is to use diffusion barriers linked to the
silica by redox sensitive bonds, typically S-S bonds, as it is
generally accepted that the redox potential as much lower in
intracellular compartments than in the extracellular milieu.
In this case, the stimuli-response under biological conditions
is built upon linker bond-cleavage by cell-produced reducing
agents. The first demonstration of redox-sensitive stimuli-
responsive cargo release based on MSNs was the capping of
the mesopores by CdS nanoparticles, where the nanoparticles
were anchored to the silica surface by a silane containing an
S-S bond in its structure [108]. As a demonstrator,
vancomycin- and adenosine triphosphate (ATP) was loaded
into the mesopores followed by CdS capping. Release of
cargo occurred upon reduction of the S-S bond. Similar
results were later reported using other nanoparticles for
capping of the mesopores, like gold [109, 110] and iron
oxide [111]. Other redox-responsive systems utilizing the
redox sensitivity of the S-S bond include the grafting of a
disilane-disulfide compound preferentially to the outer sur-
face of mesoporous silica [112] or cross-linking of a poly(N-
acryloxysuccinimide)-grafted mesoporous silica using a
disulfide-based bifunctional primary amine [113]. Layer-by-
layer assembly of oppositely charged polyelectrolyte
multilayers followed by S-S crosslinking has been also
recently demonstrated to show redox-triggerable molecular
gate properties [114].
3.2.2.3. Stimuli-Responsive Systems Based on Enzymatic
Degradation
Enzymatic degradation is a very attractive route towards
promoting intracellular release, albeit to date only demons-
trated under “in sink” conditions. The first demonstration of
an enzyme-triggered MSN-based release system was based
on the attachment of a 2-rotaxane onto the silica surface and
1176 Current Drug Targets, 2011, Vol. 12, No. 8
an ester-linked α-cyclodextrine stopper [115]. The ester bond
could then be cleaved by the use of porcine liver esterase,
which led to release of entrapped dye from the mesopores.
On a related note, α-cyclodextrine was also detached by α-
amylase-induced hydrolysis of the ester bond, leading to fast
release of cargo [116]. Another demonstration of an enzyme-
responsive triggered release system is the attachment of
amidine-units to biotinyl-functionalized MSNs [117]. In this
case, the release of a dye used as the model cargo could be
induced by addition of trypsin. Complete release of the dye
was reached in 3 h. In another interesting development,
enzyme-triggered release has been also demonstrated for
lactose-functionalized MSNs, where the lactose was cova-
lently linked to the outer surface of the MSNs. Complete
release of cargo occurred within 3 h in the presence of β-D-
galactosidase under physiological conditions. It is clear that
enzyme-triggered release is a very promising route for site-
specific drug delivery of anticancer drugs, and one can
anticipate rapid development in this field during the next
years.
3.3. Additional Selected Diffusion Barrier Concepts
In a recent development, stimuli-responsive capping of
the pores of mesoporous silica was demonstrated (under sink
conditions) using an antibody as the capping agent [118]. It
was shown that addition of an antigen (complementary to the
antibody) induces the release of the caps, which resulted in a
clearly faster release of the entrapped cargo. Although the
capping did not appear to be as tight as in many of the
systems discussed above, the concept is very exciting, as it
can allow more specific biological release systems to be
developed.
Another very interesting concept with high biological
potential relies on “protocells”, i.e. MSN supported lipid
bilayers [119]. The protocells were prepared by fusion of
positively charged liposomes onto negatively charged
MSNs. The lipid bilayer served as a diffusion barrier for
encapsulated cargo, as demonstrated for capsin. The lipo-
some fusion process could be also used to load MSNs with
cargo, which in other cases typically is carried out in a
separate step. Thus, this approach has the advantage of being
fairly straightforward in that the number of particle
preparation steps is low, and also in that the MSN surface is
inherently biocompatible. Furthermore, it would probably be
also possible to introduce targeting ligands at the same time,
which would make such particles highly interesting for drug
delivery applications. The capability of the particles to
release entrapped cargo inside cells was demonstrated in
vitro using calcein as the model drug, and the release was
suggested to be pH-triggered in nature.
3.4. Gene Delivery Systems
Since nucleic acids alone are not able to penetrate the cell
wall, the therapeutic success of gene therapy is largely
dependent on the development of efficient delivery systems
for DNA, which is typically condensed into nanoparticles by
a cationic polymer to produce non-viral gene delivery sys-
tems [120]. There have been also some successful attempts
in using MSNs as gene delivery systems. For instance,
cationically modified MSNs were shown to be able to
complex with plasmid DNA (pEGFP-C1) and successfully
Rosenholm et al.
transfected into HeLa and Chinese hamster ovarian (CHO)
cells [121]. The positive charge was provided by second
generation polyamidoamine (G2-PAMAM) dendrimers caps,
which thus simultaneously acted as gatekeepers for the
loaded Texas red dye. Even though in this case the dye was
only used to make the MSNs fluorescently visible and hence
no release was demonstrated, this system constituted an early
MSN model for a double-delivery system able of simulta-
neously delivering drugs and genes intracellularly. Later, the
same group demonstrated successful delivery of both DNA
and chemicals into plant cells using MSNs [110]. A similar
approach was recently demonstrated by Chen et al. [87],
where they fabricated MSNs encapsulated with DOX inside
the pores, which in turn were also capped with G2-PAMAM
dendrimers but complexed with siRNAs targeted against
mRNA encoding Bcl-2 protein, which is responsible for non-
pump multidrug resistance in cancer cells. In this case also
DOX release was observed under sink conditions in a
glutathione solution, whereas release in PBS was almost
negligible; and the cytotoxic activity of loaded DOX was
also demonstrated in vitro on human ovarian A2780/AD
cells.
Other modifications used to enhance the transfection
efficiency of MSNs, as they are not as such effective in
delivering nucleic acids, have included both modified
(mannosylated [122]) and non-modified PEI [72, 123]. PEI
is a cationic polymer exhibiting the highest positive charge
density when fully protonated in aqueous solution and is thus
ideal to form nanocomplexes with negatively charged
nucleic acids. Additionally, its success in gene delivery lies
in its buffering capacity at pH 4-6 which consequently leads
to its characteristic ability to destabilize lysosomal mem-
branes and thus promote endosomal escape of the genes
[120]. This property can also lead to cytotoxicity in high
doses, but when coated onto MSNs, this cytotoxicity can be
circumvented while the high transfection efficiency of PEI
can be retained. Moreover, this cationic modification of the
MSNs was shown to enhance the cellular uptake of MSNs,
in line with the common notion that cationic nanoparticles
are internalized more effectively than negatively charged
(connected to the fact that cellular membranes are negatively
charged). This is in accordance with the observation of the
uptake of amino-functionalized co-condensed MSNs as
compared to MSNs functionalized by surface-hyperbranch-
ing polymerization of PEI, where negligible uptake was seen
after 2 h incubation with HeLa cells of the co-condensed
MSNs while considerable uptake was seen for the PEI-
MSNs at the same time point [86]. Also for G2-PAMAM
dendrimers functionalized core-shell magnetite-MSNs the
uptake in glioma cells was more efficient for G2-PAMAM-
MSNs (at both 1 h and 4 h) than when these were esterified
to yield a lower surface charge of the particles [124].
3.5. MSN-Based Imaging Systems
Of the bioimagible MSN systems presented in literature,
multifunctional SPIO-based nanodevices are of particular
interest for cancer diagnosis and therapy [125] as diagnostic
tools, e.g., for early tumor detection. Other applications that
would greatly benefit of efficient in vivo imaging contrast
agents include cellular tracking. Methods that monitor the
fate and distribution of transplanted stem cells non-inva-
Multifunctional Mesoporous Silica Nanoparticles Current Drug Targets, 2011, Vol. 12, No. 8 1177

sively are for instance a key issue for developing successful
stem cell therapies [126]. MRI is an ideal imaging modality
for the biodistribution of magnetically labeled cells, whereby
in the case of MSNs, either SPIO nanoparticles [34, 127-
129] or paramagnetic gadolinium (Gd)-complexes [130-133]
can be encapsulated into the particles to render them MRI
active. Optical imaging techniques can be also utilized,
whereby the MSNs should comprise optically active func-
tions such as fluorescent dyes [134, 135] or near infra-red
(NIR) contrast agents [136]; as well as combinations of
optical and magnetic properties to provide multimodal
imaging [23, 125-128, 130, 137, 138]. While fluorescence is
an invaluable tool for the in vitro biological evaluation of
MSNs, the in vivo applicability of fluorescent (only) MSNs
is limited. A prerequisite for successful in vivo imaging
include a stable signal and in the case of cellular tracking,
that the contrast agents (in this case, the MSNs) remain
internalized long enough to be monitored. On this topic,
Mou and co-workers have investigated the influence of both
surface charge [139] and size [140] on as well as the route
[133] of the cellular uptake of MSNs. On the MRI side,
incorporating contrast agents into MSNs benefits in the sense
of extended half-lives (in the case of Gd-based contrast
agents) and enhanced cellular uptake/ labeling efficiency (in
the case of SPION) provided by the silica layer, which
furthermore can be easily further functionalized as well as
potentially be used to carry therapeutic cargo – carrying
MSNs into the burgeoning field of theragnostics.
4. MSNS AS THERAGNOSTIC AGENTS IN CANCER
4.1. MSNs in Cancer Medicine
Successful combat against cancer requires: 1) early
detection of cancer or malignant lesions; 2) identification of
the biomarkers and oncogenic signals fueling cancer prog-
ression for the choice of therapy; and, 3) efficient delivery of
the drug(s) of choice to the lesion to enhance therapeutic
efficacy and reduce unwanted side effects. MSNs meet these
demands by their ability to carry different types of
therapeutic and diagnostic entities, even simultaneously,
their high drug load capacity, the possibility for tunable
load/release mechanisms and their chemical flexibility,
allowing for modifications of size, charge and surface func-
tionalizations which determine their stability, pharmaco-
kinetics, and biodistribution (see Fig. 3). In addition to
improved therapy, this nanoplatform will offer diagnostic
benefits through targeted delivery of imaging agents for
enhanced signal detection. Further, an interesting application
is the possibility to utilize MSNs for targeted delivery of
signaling/biomarker sensors to enhance the detection of the
normally weak oncogenic bioactivity. As this drug delivery
platform can carry multiple entities they provide excellent

Fig. (3). Schematic presentation of the topics of this review addressing the aims of nanomedicine for improved cancer therapy and diagnosis
(blue arrows), the specific characteristics of mesoporous silica particles that make them especially suitable as tools to meet these aims (red
arrow to the right) and the existing challenges (red arrow to the left), when it comes to detailed understanding of the biobehavior of the drug
delivery platform that need to be attended, before this system can be considered for clinical application.
1178 Current Drug Targets, 2011, Vol. 12, No. 8
tools for multidrug delivery and combined diagnostic and
therapeutic action (theragnostics). Despite recent advances
highlighting MSNs as promising drug delivery platforms, the
development of MSNs for cancer therapeutics and diag-
nostics is still at an early stage and MSNs for targeted cancer
therapy has yet to enter clinical trials. The great challenge for
translation of this technology into medical applications is to
understand the biological behavior of MSNs.
Biology presents complex problems for material develop-
ment. Particle size, charge and surface functionalizations are
critical parameters that clearly influence the biodistribution
and pharmacokinetics of drug delivery systems. Upon
administration and before reaching the site of action the drug
delivery system will encounter a multitude of biomolecules,
cellular components and biological membranes. The system
needs to stay intact, preferentially exhibit zero premature
release of the carried therapeutics, and show minimal
interaction with and impact on the functions of biological
systems other than the targeted tissues. Interactions with
healthy cells may disturb their normal functions and lead to
vehicle-induced side effects. Protein adsorption to the drug
carriers may affect recognition of the particles by the
immune system and influence particle life time in the
circulation and toxicity. Protein coverage of the particle may
also cover active targeting molecules on the surface and
hinder recognition. The clearance mechanisms for each drug
delivery system need to be clarified to avoid problems due to
long term accumulation in healthy organs. Detailed under-
standing on how modifications/functionalizations influence
the degradation rate of the particles will be necessary to
optimize the life time of the drug delivery systems according
to the application. The rate of biodegradation, necessary for
the elimination from the body, should be matched to the
circulation time needed for the drug delivery vehicle to carry
out its function. Longer circulation times will facilitate
efficient accumulation in the target tissues whereas a more
rapid degradation will be beneficial for diagnostic action, as
that will reduce unspecific background signal from non-
targeted tissue. Biodistribution of delivery vehicle upon
intravenous administration will be also affected by the
vascular integrity and the degree of vascularization of organs
and tumors in particular. Active targeting to tumors requires
detailed knowledge of the protein make-up of the cancer
cells and the molecular function of the targeted molecule.
Due to the heterogeneous nature of cancer, it is difficult to
envision the discovery of a universal targeting ligand but
rather the optimal delivery vehicle will need to be a generic
system where ligands can be easily interchanged, based on
"targeting diagnostics" of each tumor. Detailed under-
standing on how size, charge and different surface modifica-
tions influence interactions with the immune surveillance
system, molecular and cellular functions of interacting
tissues, biodistribution and clearance from the body is
needed before the particles can advance to the clinic. Below
we discuss recent advances within MSNs research and how
MSNs can meet the above mentioned challenges for
successful application in cancer medicine.
4.2. Biocompatibility of MSNs
Safety and toxicity of the drug delivery vehicle is of
central importance and has been addressed primarily for
Rosenholm et al.
MSNs on isolated cells. Several in vitro studies demonstrate
that differently modified MSNs in the size range of 100-200
nm are generally well tolerated by several cell types at
different concentrations (typically up to 0.1 mg/mL) [141-
144], whereas unmodified MSNs show higher toxicity [145].
The influence of size, shape, and surface-charge and -
functionalizations on cellular toxicity has been discussed by
the authors in a recent review [146]. As this section deals
with medical applications we will concentrate on the in vivo
demonstrations of biocompatibility and the readers are
referred to the other review for more detailed information on
how particle characteristics influence cellular toxicity. In line
with several in vitro investigations, a recent study showed
that non-functionalized mesoporous silica materials of
different size where lethal when injected intravenously and
intraperitoneally, whereas no lethality was observed when
the particles where injected subcutaneously [147]. Post-
mortem analyses indicated that death might have been due to
thrombosis. Although subcutaneous injections did not lead to
death, they did cause severe systemic toxicity. The authors
concluded that the toxicity might be mitigated by modifica-
tions of the materials. Indeed, several in vivo studies using
functionalized MSNs have revealed no signs of acute toxi-
city in animals [147-149]. Further, a recent report demons-
trated lack of acute toxicity within 6 h after intravenous
injections of positively charged mesoporous silica nano-
particles in the size range of 50-100 nm [148]. These part-
icles were shown to be targeted to the liver but the prolonged
effect of particle administration was not addressed in this
study. Non-porous silica particles (mean size: 50-200 nm)
were recently shown to undergo renal and hepatic clearance
(urine and bile) over time in a size dependent manner [148].
Smaller particles were preferably cleared, whereas larger
particles were trapped by macrophages and accumulated in
the liver and spleen where they remained up to 4 weeks after
injection [148]. Subsequent imaging of the major organs also
further supported that the particles mainly accumulated in
the liver, even though some particles were observed also in
kidneys, lung, and spleen.
Although the studies described above did not address
long term toxicity of accumulated particles and detailed
understanding on how size, charge and surface modifications
of MSNs influence in vivo toxicity and biodistribution is still
lacking, they suggest that these drug delivery platforms
exhibit sufficient biocompatibility to be suited for
biomedical applications.
4.3. Diagnostic Applications
Medical imaging rely on positron emission tomography
(PET) and MRI. The latter is a highly sensitive technique for
the detection of ultrastructural changes, whereas PET offers
the detection of a biological activity. Early detection of the
lesion is important for a more successful treatment. The
biological signal in early lesions is normally weak and small
tumors might go undetected due to lack of specificity or poor
sensitivity. Hence delivery of imaging agents to the site of
the lesion to enhance the signal and increase specificity is of
key interest. MSNs are flexible imaging agents that can be
conjugated to imaging agents suitable for PET, MRI and
fluorescence imaging (see above). Although extremely
useful for quantification of biological behavior in vitro
Multifunctional Mesoporous Silica Nanoparticles
[150], fluorescent dyes are not the preferred option for
clinical imaging due to problems of tissue autofluorescence
and deep tissue photon penetration.
4.3.1. Magnetic Resonance Imaging
On the scarcity of in vivo studies on MSNs, most can be
found within papers dealing with imaging. Thus, most in
vivo MSN evaluations rely on MRI techniques, often applied
for core-shell type particles consisting of a magnetic core
and a mesoporous shell. For instance, Mou and co-workers
investigated the long-term applicability/contrast enhance-
ment of their developed tumberlike so-called Mag-
Dye@MSNs [123], with both optical (fluorescent) and
magnetic properties, both after intravenous administration
via eye vein injection [125], and implantation of Mag-
Dye@MSN-labeled human mesenchymal stem cells
(hMSCs) at the olfactory cortex of the brain [124] by MRI.
In the previous case, the Mag-Dye@MSNs were found to
accumulate into organs belonging to the reticuloendothelial
system (RES), mainly spleen and liver. They were able to
monitor the liver for 3 months, whereby it was found that the
signal-to-noise ratio recovered after 90 days, indicating that
the Mag-Dye@MSNs were resistant to decomposition and
not easily excreted from the body, and thus a potential
candidate for long-term liver/spleen MRI monitoring [125].
In the latter case, the animal was imaged by MRI 9 days
after hMSC implantation, i.e. after long-term growth and
differentiation, and the Mag-Dye@MSN-labeled hMSCs
were still visualized [124]. Moreover, in this study, the
labeled cells can be visualized in a clinical 1.5 T MRI system
with an in vitro minimal detectable cell number of about
104, and an in vivo detection quantity of 1.25 × 105 cells,
whereas in the former study a 7 T MRI was used for animal
imaging. Hyeon and co-workers also applied dually active
(fluorescent and magnetic) PEGylated MSNs and examined
their passive tumor accumulation after intravenous injection
as well as visibility after subcutaneous injections of MCF-7
cells labeled with Fe3O4@mSiO2 [34]. After 2 and up to 24 h
post-injection, the accumulation of the MSNs was detected
in the T2-weighted MR images. Recently, the same group
repeated a similar protocol but based on a MSN system with
surface-bound Fe3O4 nanocrystals, also rendering them
magnetically active for MRI [34]. Also these MSNs were
passively targeted to shoulder-implanted MCF-7 tumors.
Thus, they also attempted to deliver DOX to the tumor sites
with the aid of the MSNs as delivery vehicles, and were
indeed able to observe DOX-originating fluorescence as well
as apoptosis in the harvested tumor cells 48 h post-injection
sacrifice. However, since no free DOX was used as control,
it remains slightly unclear if drug delivery was in fact
particle-mediated or due to free (leached) drug. However,
this study constitutes a promising step towards the use of
MSNs as theragnostic agents.
MSNs have also shown to be feasible candidates as T1
contrast agents. For instance, Mou and co-workers have
fabricated MSNs tagged with both the FITC fluorophore and
a paramagnetic T1 contrast agent, Gd-DTPA, and implanted
Gd-Dye@MSN-labeled hMSCs into the basal ganglions of
nude mice, which could be visualized under a clinical 1.5 T
MR system even 14 days post-implantation [130]. Likewise,
Taylor et al. [131] grafted MSNs with a Gd-DTTA (diethyl-
enetriaminetetraacetic acid) chelate. It was found that MSNs
Current Drug Targets, 2011, Vol. 12, No. 8 1179
was an ideal platform for efficient MRI due to their ability to
carry large payloads of Gd centers, along with a good water
accessibility of the Gd-chelates inside the porous matrix.
Carniato et al. [133] carried out an investigation of pore size
dependence (MCM-41, dp = 33 Å vs. SBA-15, dp = 82 Å) on
the water accessibility in terms of their 1H nuclear magnetic
resonance (NMR) relaxometric properties as a function of
temperature and magnetic field strength. They found a 4-fold
difference in the relaxivity values by the different accessi-
bility of water molecules to Gd centers localized inside or
outside the pores. Thus, the Gd-complexes grafted outside
the pores, i.e. mostly on the particle surface, contributed to
the observed relaxivity to the largest extent, an effect which
was especially pronounced for the MCM-41 structure for
which accessibility of the Gd-centers inside the pores was
more limited. However, when comparing Gd-labeled MCM-
41 to the commercial contrast agent ProHance®, the former
phantoms (3.0 T) showed a remarkable 1H R1 enhancement
at increasing Gd concentrations compared to ProHance®
phantoms. Also in vivo, Taylor et al. [131] found that much
smaller doses of their Gd-MSNs were needed as compared to
typical Gd doses for a significant T1-weighted enhancement
(9.4 T) of the aorta of a DBA/1J mouse 15 min post-injection
into the tail vein. They concluded that Gd-MSNs could be
suitable for intravascular MRI. In line with the findings of
Mou and co-workers described above, after injecting higher
doses into the tail vein, a significant loss of MR signal was
observed in the liver 1 h post-injection. This indicated that
Gd-labeled MSNs could be used as contrast enhancers also
for T2-weighted MR images. They postulated that liver
signal loss was a result of phagocytosis of the particles by
liver macrophage cells, which they demonstrated in vitro
with related monocyte cells in the same study [131].
4.3.2. Biosensing
Detection of biological activity is an attractive diagnostic
application [149, 150]. The large surface area of MSNs
allowing for conjugation with a high concentration of sen-
sing molecules, and the optical transparency which permits
high detection rate of the signal, render MSNs highly suita-
ble for biosensing. Drugs, enzymes, antibodies, and nucleo-
tides, all suitable for MSNs incorporation, can serve as
recognition systems in biosensing applications. Although not
demonstrated for in vivo activity, MSNs have been
successfully synthesized for the detection of glucose [151],
H2O2 [152], and NO2 [153]. An example of a molecular
recognition system capable of distinguishing between struct-
urally similar neurotransmitters utilizing different functiona-
lizations of the exterior and the interior particle surfaces was
shown by Radu et al. [154]. The functionalizations affected
the diffusion rate, and hence detection of the analytes into
the particles based on their charge. Martínez and co-workers
developed a MSN based system for the recognition of anions
that were capable to respond to different degrees to AMP,
adenosine diphosphate (ADP), and ATP [155]. Although the
above described systems have been not applied for cancer
diagnostics, they demonstrate the flexibility of the tech-
nology platform and the potential for MSNs in biosensing.
MSN-mediated delivery of signaling sensors or antibodies
recognizing specific biomarkers for cancer might provide an
additional value in the diagnosis of the oncogenic activity,
and facilitate the choice of therapeutic modality.
1180 Current Drug Targets, 2011, Vol. 12, No. 8
4.4. Therapeutic Applications
4.4.1. Enhanced Therapeutic Efficacy through Efficient
Drug Delivery
The therapeutic window of many drugs is very narrow
due to frequent occurrence of serious side effects, arising
from a non-specific drug uptake by healthy cells. Further
therapeutic efficacy of many drugs is hampered by low
active concentrations due to poor drug solubility, drug
instability, and poor cellular uptake of the active agent.
Therefore, the development of delivery systems that can
carry a high drug payload, protect the drug from degradation,
target specific cell populations, and facilitate cellular uptake
of the active agent is necessary for clinical applicability of
many drugs. Mesoporous silica structures has been utilized
for intracellular delivery of both hydrophobic anticancer
agents such as camptothecin [135, 156] and paclitaxel [70,
157, 158], and the more hydrophilic DOX [34, 120]. Due to
the enhanced cellular uptake of MSNs (and hence the more
efficient drug delivery), the intracellular drug concentrations
and the subsequent biological effects were enhanced. Such
enhanced and furthermore selective drug action by MSN-
mediated drug delivery was also recently shown, where the
level of apoptosis induced by MTX-conjugated nanoparticles
on cancer cells was significantly higher than that of corres-
ponding amounts of free drug, whereas the non-cancerous
cell line remained unaffected. Additionally, successful deliv-
ery of nucleic acids [70, 117, 121, 159] highlights the
potential of MSNs in gene therapy. The high drug load
capacity and flexible drug coupling chemistry of MSNs also
permits multidrug delivery, which is a critical issue in cancer
therapy as tumors exhibit a complex cross talk between
oncogenic signals and often develop resistance to a given
drug. Rosenholm et al. recently showed successful co-
delivery of two hydrophobic model drugs, the fluorescent
molecules 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbo-
cyanine perchlorate (DiI, red color), and 3,3'-dioctadecyloxa-
carbocyanine perchlorate (DiO, green color), which were
simultaneously loaded into the pore network, further
emphasizing the potential for multidrug delivery [86].
Despite the large number of recent reports demonstrating
the capability of MSNs to deliver drugs under in vitro con-
ditions, the therapeutic efficiency of drug-containing MSNs
has been not demonstrated under in vivo conditions. To the
best of our knowledge, the only in vivo study involving bio-
logically active mesoporous silica is the recent investigation
by Dai et al. [160]. They showed that mesoporous silica
doped with calcium and silver significantly activate the int-
rinsic pathway of coagulation cascade, induce platelet adher-
ence, promote the blood clotting, and achieve hemorrhage
control in femoral artery and liver, when administered as a
powder form directly onto an open wound in a rabbit model.
In an earlier study, Mellaerts et al. loaded the poorly water-
soluble drug itraconazole into mesoporous silica to enhance
the oral availability of the drug [161]. However, neither of
these studied were focused on nanoparticulate formulations.
4.4.2. Fighting Multidrug Resistance
Multidrug resistance is a major obstacle in cancer
chemotherapy and severely limits the efficacy of cancer
therapeutics. Both cell intrinsic and extrinsic mechanisms
can lay behind this property. Cellular mechanisms that affect
Rosenholm et al.
drug resistance include induction of cellular repair systems,
reduced apoptotic sensitivity, and/or increasing drug efflux
in tumor cells. The latter, also denoted as pump mechanisms
of drug resistance, is mediated by membrane glycoproteins
that function as drug transporters. Nanoparticles can bypass
pump mechanisms by efficient intracellular drug delivery
which allows for obtaining high intracellular drug concentra-
tions, counteracting the activity of multidrug resistance
pumps [149]. Other cellular resistance mechanisms can be
overcome by specific targeting of the responsible molecules.
Such an approach was recently demonstrated by Chen et al.
[87]. They used DOX-loaded MSNs carrying siRNAs tar-
geted against the antiapoptotic Bcl-2 protein. The MSN
complex was delivered into multidrug-resistant A2780/AD
human ovarian cancer cells to induce significantly enhanced
cell death, compared to free DOX. They attributed this effect
to the knockdown silencing of Bcl-2 mRNA, leading to
enhanced cytotoxicity of the drug. Recently, it has been
reported the successful delivery of a siRNA construct cap-
able of knocking down GFP expression on PANC-1, BxPC3,
and HEPA-1 cell lines [87]. Moreover, thanks to the en-
hanced cellular uptake of the cationically modified MSNs,
the delivery of paclitaxel to the pancreatic cancer cell line
(PANC-1) was increased. This study postulated the simulta-
neous delivery of siRNA (to knock down the expression of
the P-glycoprotein drug exporter) and chemotherapy agents
that are actively exported by this glycoprotein, as an
effective strategy in the treatment of drug resistant cancers
based on pump mechanisms. Thus, the multimodality of
MSNs allows for intelligent design in providing synergistic
capabilities, which could be also utilized to overcome both
pump and non-pump multidrug resistances in cancer cells
(see Fig. 4).
Poor vascularizations that restrict access of the drug
delivery system to the tumor site, high interstitial pressure
and low microvascular pressure counteracting extravasation
of the drug carrier from the tumor mass comprise cell
extrinsic mechanisms of drug resistance. Such mechanisms
are harder to overcome especially for larger vehicles. They
may require local instead of intravenous administration,
combined with mechanisms that enhance cellular interact-
tions and uptake.
4.4.3. Targeted Therapy
A core problem of current anticancer drugs is the very
high incidence of severe unwanted side effects in healthy
tissues. The ultimate goal in cancer therapy is to restrict the
therapeutic effect exclusively to cancerous cells whereas the
healthy tissue should be minimally affected. Nanoparticles
have taken the center stage as drug carriers for targeted
delivery. Tumor targeting can be principally achieved
through the combined effect of passive targeting and active
targeting strategies. Due to the leaky vasculature and poorly
operational lymph system of tumors, nanoparticles accu-
mulate into the tumor site by passive targeting, i.e. via the
enhanced permeability and retention (EPR) effect, whereas
the function of active targeting is to facilitate the interaction
with cancer cells, retain the vehicle into the tumor site, and
facilitate the cellular uptake [15]. MSNs have been con-
jugated with various targeting ligands including sugar
moieties [121, 162, 163], folic acid [164, 165], DNA
aptamers [167], as well as monoclonal antibodies, e.g.,
Multifunctional Mesoporous Silica Nanoparticles
Endosome
Cytotoxic action
Nucleus
Antiapoptotic mechanisms
Drug resistance mechanisms
Fig. (4). MSNs can be designed to overcome multidrug resistance mechanisms by increasing the intracellular concentration of chemotherapy
agents as well as silencing the synthesis of proteins involved in drug resistance to enhance the effect of the loaded cytotoxic drug. Adapted
from ref. [122].
against the human epidermal growth factor receptor 2
(HER2) [166]. Successful cell specific drug delivery to
cancer cells have been demonstrated by Zink and co-
workers. They showed elevated cytotoxicity in pancreatic
PANC-1 cancer cells (60 % cell death based on a viability
assay) by MSNs surface functionalized with folate and
loaded with the anticancer drug camptothecin, as compared
to the HFF cell line (30 % cell death) used as reference cell
line [135]. Similar numbers were reported by Zhu et al.
[114], who investigated the effect of DOX-loaded MSNs
surface functionalized with aptamer conjugates on cell
viability in cancerous HeLa cells and healthy QGY7703
cells. About 40 % and 60 % viability was reported after 24 h,
respectively. In a recent study, Rosenholm et al. [83] con-
jugated the anticancer drug MTX, structurally similar to
folate, mainly onto the surface of MSNs. MTX acted dually
as a targeting ligand and a cytostatic drug. The MTX-loaded
MSNs induced an apoptotic cell death of about 33 % after 72
h in the cancerous HeLa cell line, compared to non-can-
cerous HEK293 cells where no apoptosis over the control
was observed.
We are still awaiting demonstrations on the ability of
MSNs for targeted drug-delivery in vivo, but the above
mentioned studies and the fairly straightforward functiona-
lization of MSNs for targeting purposes make them pro-
mising candidates for targeted drug delivery.
4.4.4. Photodynamic Therapy
A recent development in the therapeutic application of
MSNs is the attachment of cell-death-inducing photosensi-
tizes in the pores for photodynamic therapy (PDT) [162, 167,
Current Drug Targets, 2011, Vol. 12, No. 8 1181
Drug
siRNA
Targeting moiety
Targeted receptor
Cell specific apoptosis
168]. PDT involves the administration of photosensitizers
(PS) with subsequent irradiation of appropriate wavelengths
to induce cell death. The overall reaction generates free
radicals and reactive oxygen species, such as singlet oxygen
(1O2), which are toxic [162]. Light acts like a trigger to
induce toxicity, causing subsequent cell damage or even
death. The large surface area of MSNs thus facilitates a high
loading of PS while the outer particle surface can be tailored
for efficient cellular uptake or even targeting [168]. More-
over, the ability of PS to enter cells is critical, since the
short-lived 1O2 can then directly interact with intracellular
machinery for maximal cytotoxicity and thus when
conjugated inside the pores of MSNs, the PS remain well-
protected from its local environment within a rigid porous
structure. Mou and co-workers thus reported a surface
modification process conjugating a PS, protoporphyrin IX
(PpIX) [167] as well as a Pd-porphyrin (PdTTP) [168], with
MSNs through covalent bonding. In vitro evaluation of
PdTTP-MSNs revealed significant photo-induced cyto-
toxicity as studied in a MDA-MB-231 breast cancer cell line,
and furthermore combined the ability of PdTPP to serve as
an in vivo contrast agent for oxygen sensing and imaging.
The team concluded that PdTTP-MSNs holds great promise
as a useful nanoplatform for cancer theragnostics [168]. In
vitro tests of PpIX-MSN performed with HeLa cells also
revealed high cellular-uptake efficiency, and the
phototoxicity was found to be both irradiation time- and
dose-dependent, while cytotoxicity would occur only in the
irradiated area. They thus concluded that small amounts of
PpIX-MSNs accumulated inside healthy cells are fairly
tolerable and would not cause severe cell damages to unra-
1182 Current Drug Targets, 2011, Vol. 12, No. 8 Rosenholm et al.

diated regions. This would be beneficial in cancer treatment, DOX = Doxorobucin

as the resulting photodynamic effect can then be limited to
EDC = 1-Ethyl-3-(3-dimethylaminopropyl)-
the area of interest, leaving the surrounding healthy tissues
carbodiimide
and cells undamaged. Another strategy to accomplish this is,
naturally, active cellular targeting of the cancer cells. In this EPR effect = Enhanced permeability and retention effect
way, a recent report by Brevet et al. designed mannose- Gd = Gadolinium
targeted MSNs for PDT and showed that targeting of also
MDA-MB-231 cells with mannose was essential (as GFP = Green fluorescent protein
compared to non-functionalized MSNs) to get a high PDT HER2 = Human epidermal growth factor receptor 2
efficiency in this case [162].
hMSCs = Human mesenchymal stem cells
MRI = Magnetic resonance imaging
OUTLOOK
Fe3O4 = Magnetite
MSNs have been shown during the last ten years to
exhibit numerous attractive properties that could be syner- MIMs = Mechanically interlocked molecules
gistically exploited in the development of theragnostic MSNs = Mesoporous silica nanoparticles
devices for targeted medicine. On the therapeutic side,
MSNs have proven to function as delivery vehicles for a MTX = Methotrexate
range of different drugs, proteins, and nucleic acids, and MCM-41 = Mobil composition of matter No. 41
have also recently demonstrated successful multidrug deli-
very potential. Different cargo release strategies can be built NIR = Near infra-red
into the system, which can be based on either simple diffu- NMR = Nuclear magnetic resonance
sion that can be tuned by the material characteristics, or more
sophisticated mechanisms based on either intrinsic (release PdTTP = Pd-porphyrin
triggered by intracellular changes in pH, redox potential, or PBS = Phosphate buffered saline
enzymes) or external (irradiation, magnetization, ultrasound,
PDT = Photodynamic therapy
temperature) triggers. Hereby caps consisting of other
nanomaterials, mechanically interlocked molecules (MIMs), PS = Photosensitizers
proteins (even antibodies) or supramolecular assemblies can
PAH/PSS = Poly(allylamine) hydrochloride/
function as gate-keepers locking the drug molecules prior to
poly(sodium-4-styrene sulfonate)
exposure to a trigger. Also stimuli-responsive polymeric
chains can be utilized for this purpose, all of the former exhi- PEG = Polyethylene glycol
biting some true synergistic capabilities of hybrid inorganic- PEI = Polyethyleneimine
organic materials. On the imaging side, MSNs have shown
to be suitable for multimodal imaging, to date demonstrated PET = Positron emission tomography
under both in vitro and in vivo conditions. Due to their PLGA = Poly(D,L-lactide-co-glycolide)
multifunctionality, these imaging properties can be readily
combined with the drug delivery ability to yield interesting PNIPAAm = Poly(N-isopropylacrylamide)
theragnostic agents. Furthermore, as targetability has re- PpIX = Protoporphyrin IX
cently been successfully demonstrated for these systems,
they contain all elements for the design of smart theragnostic RES = Reticuloendothelial system
nanoplatforms. Even though the therapeutic benefits of G2-PAMAM = Second generation polyamidoamine
MSNs in vivo remains to be demonstrated, emerging bio-
compatibility and biodistribution studies together with the SBF = Simulated body fluid
promising in vitro outcomes suggest in the proximate future 1O2 = Singlet oxygen
the expansion of MSNs into such in vivo applications.
SPIO = Superparamagnetic iron oxide
TEM = Transmission electron micrographs
ABBREVIATIONS
XRD = X-ray diffraction
AMP = Adenosine monophosphate
ADP = Adenosine diphosphate REFERENCES
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CTAB = Cetyltrimethylammonium bromide nanoparticles deliver? Drug Discov Develop 2009; 12: 30-4.
[2] Sahoo SK, Parveen S, Panda JJ. The present and future of
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