Targeting of Porous Hybrid Silica Nanoparticles to Cancer Cells

Article (PDF Available)inACS Nano 3(1):197-206 · February 2009with503 Reads
DOI: 10.1021/nn800781r · Source: PubMed
Abstract
Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of poly(ethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in many cancer cells as compared to normal cells, folic acid was used as the targeting ligand. The internalization of the particles in cell lines expressing different levels of folate receptors was studied. Flow cytometry was used to quantify the mean number of nanoparticles internalized per cell. Five times more particles were internalized by cancer cells expressing folate receptors as compared to the normal cells expressing low levels of the receptor. Not only the number of nanoparticles internalized per cell, but also the fraction of cells that had internalized nanoparticles was higher. The total number of particles internalized by the cancer cells was, therefore, about an order of magnitude higher than the total number of particles internalized by normal cells, a difference high enough to be of significant biological importance. In addition, the biospecifically tagged hybrid PEI-silica particles were shown to be noncytotoxic and able to specifically target folate receptor-expressing cancer cells also under coculture conditions.
Targeting of Porous Hybrid Silica
Nanoparticles to Cancer Cells
Jessica M. Rosenholm,
Annika Meinander,
‡,§,
Emilia Peuhu,
‡,§,¶
Rasmus Niemi,
John E. Eriksson,
‡,§
Cecilia Sahlgren,
‡,§,
* and Mika Linde´n
†,
*
Center for Functional Materials, Department of Physical Chemistry, Åbo Akademi University, Porthansgatan 3-5, FI-2500 Turku, Finland,
Department of Biology, Åbo
Akademi University, Artillerigatan 6A, FI-20520 Turku,
§
Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, P.O. Box 123, FI-20521, Turku,
Finland, and
Current address: The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, London SW3 6JB, United Kingdom.
Both
authors contributed equally to this work.
T
argeting and drug delivery to spe-
cific cell populations are key aims in
biomedical science today. Research
efforts are, therefore, being placed on gen-
erating drug delivery systems to specifically
target drugs to malfunctioning cells. Cellu-
lar targeting by utilizing antibodies or spe-
cific ligands rely on the capability of the tar-
geting agents to selectively bind to the cell
surface to trigger receptor-mediated en-
docytosis.
1
The drug delivery system along
with the therapeutic agent would thereby
be delivered to the interior of a given type
of cells. Such a drug delivery system will not
only have important therapeutic and phar-
macological applications but also be of
great interest for medical imaging and diag-
nosis. Targeting is especially relevant in the
context of cancer therapies, as most of the
commonly used anticancer drugs have seri-
ous side-effects due to unspecific action on
healthy cells. The selectivity is a function of
the ability of the nanoparticles to be inter-
nalized by the targeted cell population. But
at least as important for the biological activ-
ity is the efficiency of uptake, that is, num-
ber of nanoparticles internalized by cancer
cells in comparison to nontarget cells, as
drug concentration is a key parameter for
successful treatment.
The folate receptor, which normally is
expressed only at the luminal surface of po-
larized epithelia, inaccessible to the
circulation,
2,3
has been found to be overex-
pressed on the surface of several cancer
cells, such as ovarian, endometrial, colorec-
tal, breast, lung, renal cell carcinoma, brain
metastases derived from epithelial cancer,
and neuroendocrine carcinoma.
4,5
Because
of this distinguishing feature between nor-
mal and cancer cells, folic acid, FA, has
emerged as an attractive targeting ligand
for selective delivery.
6
For a nanoparticu-
late delivery device, the challenge lies in
successfully combining therapeutic and tar-
geting actions with imaging capability into
one system. In many cases, it is difficult to
couple several functional groups in suffi-
cient concentration, since the number of at-
tachment sites on the particle surface is lim-
ited.
7
Moreover, each functionalization step
might negatively affect the suspension sta-
bility of the particulate system, depending
on the physicochemical properties of the
added function.
7,8
Examples of drug deliv-
ery systems that have been successfully syn-
thesized to combine targeting, imaging,
and therapeutic moieties on their surface
are dendrimers.
911
Dendrimers are nanom-
eter sized, nonimmunogenic and hyper-
branched “starburst polymers”,
12
which be-
cause of the great number of terminal
functional groups can be efficiently tai-
lored for spatial distribution of biospecific
moieties.
13
Furthermore, as these
*Address correspondence to
Cecilia.Sahlgren@abo.fi,
Mika.Linden@abo.fi.
Received for review September 26,
2008 and accepted December 05, 2008.
Published online December 23, 2008.
10.1021/nn800781r CCC: $40.75
© 2009 American Chemical Society
ABSTRACT Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of
poly(ethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with
the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in many cancer cells
as compared to normal cells, folic acid was used as the targeting ligand. The internalization of the particles in
cell lines expressing different levels of folate receptors was studied. Flow cytometry was used to quantify the mean
number of nanoparticles internalized per cell. Five times more particles were internalized by cancer cells
expressing folate receptors as compared to the normal cells expressing low levels of the receptor. Not only the
number of nanoparticles internalized per cell, but also the fraction of cells that had internalized nanoparticles was
higher. The total number of particles internalized by the cancer cells was, therefore, about an order of magnitude
higher than the total number of particles internalized by normal cells, a difference high enough to be of significant
biological importance. In addition, the biospecifically tagged hybrid PEI-silica particles were shown to be
noncytotoxic and able to specifically target folate receptor-expressing cancer cells also under coculture conditions.
KEYWORDS: Cellular targeting · mesoporous silica ·
bioimaging · nanoparticles · surface functionalization
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functional end groups are often primary amines, the
high positive charge density provided by dendrimers
and other polycations, such as poly(ethylene imine), PEI,
are commonly applied as gene transfection agents. PEI
has been reported to offer high gene delivery efficiency,
since it exhibits a unique “proton-sponge”
14
or “endo-
some buffering” effect,
15
thus destabilizing lysosomal
membranes and promoting endosomal escape. Be-
cause of these properties, recent interest has been fo-
cused toward the use of dendrimers as multifunctional
FA-conjugated nanodevices for cancer therapy
7,9,10,16,17
and inflammatory tissue.
18
Alongside with conventional polymeric carrier
materials, ceramic structures have emerged as prom-
ising alternatives for drug delivery systems. Incorpo-
ration of drug substances into solgel derived silica
(SiO
2
) materials was introduced as early as 1983.
19
The solgel method is a simple and versatile low-
temperature way of preparing porous, amorphous
ceramic materials, which are stable toward light and
heat, without being hazardous to humans or the en-
vironment.
20
Silica is an essential component of cells
throughout the human body and amorphous silica is
known to be nontoxic, biocompatible and biode-
gradable, being freely dispersible throughout the
body and ultimately excreted in the urine. Especially
room-temperature processed mesoporous silica is
rapidly dissolved in water,
21
but when loaded with
large amounts of guest molecules such as drugs, the
aqueous solubility can be slowed down because of
the increased hydrophobicity of the pore walls.
22
This large adsorption (drug loading) capacity is one
of the many attractive features of mesoporous silica
materials, accompanied by control of material prop-
erties on the nanometer scale such as pore and par-
ticle size. The drug content per mesoporous silica
nanoparticle can be varied in a fairly straightfor-
ward fashion by optimizing the drug incorporation
conditions.
2224
The feasibility of mesoporous silica
as a drug delivery system has, thus, been docu-
mented since the beginning of the millennium and
has been subject to a number of reviews.
2430
These
studies have demonstrated examples of mesopo-
rous silica as a vehicle for successful intracellular de-
livery of otherwise poorly soluble
31,32
or membrane-
impermeable agents,
33
such as anticancer drugs.
However, as was pointed out in a very recent re-
view, cell type specificity is a challenge that still
Figure 1. Schematic representation of the hybrid silica nanoparticle structure.
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needs to be overcome for these types of materi-
als.
30
Recently, we have reported the synthesis of hybrid
PEI-mesoporous silica particles, where hyperbranched
PEI is covalently linked to the silica surface by growing
PEI via surface-polymerization.
34,35
Such hybrid particles
are suggested to combine most of the positive fea-
tures of dendrimers on the one hand, and mesoporous
silica on the other, with regard to their use as drug car-
rier matrixes. While dendrimers are prepared by syn-
thetic procedures involving iterative synthesis steps, hy-
perbranched polymers such as the PEI-function are
conveniently prepared in one step. The ceramic carrier
matrix furthermore effectively protects the payload
molecules from (e.g., enzymatic) degradation during de-
livery, as well as increases solubility and permeability
of a drug with otherwise poor bioavailability.
36
The hy-
brid PEI-mesoporous silica particles have a much higher
surface concentration of amino-groups as compared
to corresponding materials prepared by classical co-
condensation or postsynthesis grafting using amino-
silanes.
35
As the isoelectric point, IEP, of the particles ex-
ceeds 9, their dispersion stability is dramatically
enhanced as the surface maintains a high positive
charge at physiological pH.
8
This is of great impor-
tance for the prevention of aggregation under physi-
ological conditions. An additional level of flexibility of
our approach is that the surface charge of the PEI-
functionalized silica particles can be fine-tuned by
chemical modifications such as succinylation,
37
to gain
electrostatic stability under physiological pH conditions
having a net negative outer surface charge on the par-
ticles. Importantly, the covalent attachment of the PEI
layer to the particle surface ensures that the polymeric
Figure 4. Folic acid-conjugation increases nanoparticle endocytosis in HeLa
cells. HeLa cells were treated with nanoparticles at a dose of 10 g/ml for 2 h
and the extracellular binding of nanoparticles was quenched with trypan
blue (FITC, pristine FITC-labeled particle; FITC/PEI, PEI-functionalized FITC-
labeled particle; FITC/PEI/FA, folic acid-conjugated PEI-functionalized FITC-
labeled particle). The FITC-labeled particles inside the cells were imaged by
fluorescence microscopy and the cellular morphology by differential interfer-
ence contrast (DIC) microscopy (A). The number of HeLa cells with endocy-
tosed nanoparticles in control or trypan blue quenched cells was detected by
flow cytometry (B). The results are representatives of at least three indepen-
dent experiments.
Figure 5. Folic acid competition inhibits specific particle en-
docytosis. HeLa cells were cultured overnight with 03mM
folic acid (FA) after which the cells were left untreated (con-
trol) or incubated with 10 g/ml FITC/PEI/FA-functionalized
nanoparticles (nano) for 4 h. After incubation the extracellu-
lar fluorescence was quenched by trypan blue and the en-
docytosed particles with FITC label were detected by flow cy-
tometry. Mean fluorescence intensity (MFI) of FITC
fluorescence in the cells was measured and the values nor-
malized to particle endocytosis of HeLa cells (n 2; mean
SEM).
Figure 2. SEM image of APS-functionalized mesoporous
silica nanoparticles.
Figure 3. -Potential titrations for mesoporous silica par-
ticles before (9) and after () PEI-functionalization, as well
as after FITC- and FA-conjugation (). Dotted line at physi-
ological pH indicates low electrostatic suspension stability
for nonpolymer-modified particles.
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surface function remains on the particle throughout ad-
ditional functionalization steps as well as during appli-
cation. Our system thus carries similarities with a triple
coreshellshell architecture: an outer shell with one
functionality such as targeting, an inner core consisting
of a biodegradable mesoporous silica matrix able of car-
rying, protecting, and releasing large amounts of cargo;
along with a middle PEI-layer which should enhance
suspension stability, promote endosomal escape (see,
for example, the recent study by Fuller et al.
38
)aswellas
provide reactive “hooks” that enable further modifica-
tions by standard bioconjugation routes.
To validate the applicability of our hybrid system in
cancer cell-targeting applications, mesoporous nano-
particles with a mean diameter of about 400 nm were
prepared and further functionalized with surface hyper-
branching polymerization of PEI. This particle size was
chosen since smaller particles typically show enhanced
unspecific cellular uptake. For example, Lin et al.have
shown that surface-functionalized 100 nm silica par-
Figure 6. FITC/PEI/FA-functionalized silica nanoparticles do not induce cell death. HeLa cells were incubated with 2 g/ml
(A) or 10 g/ml (B) of nanoparticles for 24 h. The endocytosed particles with FITC label (green) and DAPI-labeled cell nuclei
(blue) were detected by confocal microscopy. Alternatively the cells were disrupted and the nuclei labeled for DNA content
with propidium iodide. The samples were analyzed by flow cytometry and the fraction of sub-G0/G1 events was detected as
a measure of apoptotic cell death (C). The relative fluorescence intensity of propidium iodide per cell was measured at the
FL3-H channel. For a positive control the cells were treated with 1 M staurosporine (STS). The results are representative of
two independent experiments.
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ticles are effectively internalized by
HeLa cells regardless of the surface
composition of the particles.
39
More-
over, for amorphous silica, toxicity
tends to be inversely related to par-
ticle size, and a particle size below 100
nm has actually been found to induce
cytotoxicity in some cases.
40
A fluores-
cent dye, in the present case FITC,
was covalently linked to the outer sur-
face of the particles, making them vis-
ible by fluorescence microscopy and
flow cytometry. Folic acid was used as
the targeting ligand, and the cancer-
specific internalization of these par-
ticles was tested on tumor and nontu-
mor originating cells. HeLa cervical
carcinoma cells were used as model cancer cells, as
HeLa cells express the folate receptor- (FR-).
41
The
embryonic kidney epithelial 293 cell line was used as a
“folate receptor-negative” noncancerous cell model as
the concentration of FR- is much lower in these cells as
compared to the HeLa cells. Importantly, the extracellu-
lar FITC was quenched with trypan blue (TB) in order
to distinguish between the particles attached to the
membrane and the particles internalized by the cells.
Flow cytometry was used for the quantification of the
fraction of cells that had internalized nanoparticles as
the percent (%) of FITC positive cells among the given
number of cells counted, as well as for measuring the
mean quantity of nanoparticles internalized per cell as
the mean fluorescence intensity among the counted
cells. Even though investigations regarding cytotoxic-
ity of mesoporous materials can be found in the litera-
ture (see for example refs 4244), cytotoxicity seems to
be particle-specific, and therefore cytotoxicity assays
were also conducted. The endocytosis mechanism was
verified by competing experiments with free folic acid,
and the targetability was finally confirmed under cocul-
ture conditions.
RESULTS AND DISCUSSION
Nanoparticle characterization. The investigated nanopar-
ticulate system is schematically shown in Figure 1, and
an SEM image of the particles is shown in Figure 2. Par-
tially aminofunctionalized mesoporous silica nanoparti-
cles with a mean diameter of about 400 nm were first
synthesized using a mixture of aminotrimethoxysilane
(APS) and tetramethoxysilane (TMOS) as the silica pre-
cursors and cetyltrimethylammonium chloride (CTACl)
as the structure directing agent. The particles had an or-
dered arrangement of mesopores, a specific surface
area of 850 m
2
/g, a pore diameter of about 3.5 nm, and
a pore volume of 0.6 cm
3
/g (see Supporting Informa-
tion). The corresponding values after PEI-
functionalization were 720 m
2
/g, 2.8 nm, and 0.34
cm
3
/g, respectively. The PEI content of the hybrid silica
particles was 18 wt % as determined by thermogravim-
etry and the particle size distribution was measured by
dynamic light scattering, indicating that no particle ag-
gregation had occurred during functionalization (see
Supporting Information). The particles were then fluo-
rescently labeled by covalently attaching FITC contain-
ing the amine specific NACAS group to primary
amine groups on the surface-grown PEI or
aminopropyl-groups in the case of nonpolymer-
functionalized particles. Finally, the targeting ligand,
folic acid, FA, was covalently attached to amino groups
through carbodiimide coupling. The surface modifica-
tions can easily be followed by -potential measure-
ments, which are sensitive mainly to the outer surface
of the nanoparticles. As seen in Figure 3, the isoelectric
point, IEP, of the silica nanoparticles increased from 7
for the starting particles, to 9.6 upon PEI- functionaliza-
tion, reflecting the pronounced increase in the surface
amino group density. The -potential titration curve of
the PEI-functionalized particles did not show any hys-
teresis, demonstrating the high chemical stability of the
PEI-silica linkage. The IEP remained virtually constant
upon FITC conjugation, but decreased slightly but sig-
nificantly upon conjugation of the acidic FA to the FITC/
PEI particles, from 9.5 to 8.7, as expected (see Figure
3). Thus, the changes in the IEP of the particles sug-
gest that the surface conjugations of PEI, FITC, and FA
to the mesoporous silica particles were successful. The
-potential value at physiological pH of 7.4 was 30
mV, which makes the particles fully dispersible in wa-
ter, in agreement with our previous study.
8
Importantly,
the -potential value, and therefore also the dispersabil-
ity, remained unchanged also at physiological pH in a
HEPES buffer solution, which is typically used for buffer-
ing cell media.
Cellular Uptake As a Function of Particle Surface Modification.
The uptakes of the plain (FITC), FITC/PEI- and FITC/PEI/
FA-functionalized nanoparticles by HeLa cancer cells
were studied by fluorescence microscopy and flow cy-
tometry under in vitro conditions. The HeLa cells were
Figure 7. Folate receptor surface expression. HeLa cells and 293 cells were trypsinized and
labeled with antifolate receptor primary antibody followed by Alexa488-conjugated sec-
ondary antibody (black line). Nonspecific fluorescence was measured using the secondary
antibody only (shaded area). The samples were analyzed by flow cytometry by measuring
the relative fluorescence intensity of FITC per cell at the FITC-A channel. The results are rep-
resentative of two independent experiments.
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incubated with nanoparticles for2hat3Catadose
of 10 g/ml. As shown in Figure 4B, flow cytometry
measurements indicate that about 70% of the cells
showed enhanced fluorescence when incubated with
PEI-functionalized particles. As these particles carry a
highly positive surface charge under the studied condi-
tions, electrostatic attraction of the particles to the
negatively charged cell membrane (50 mV for HeLa
cells) could account for this observation. To reliably dis-
tinguish between particles just attached to the outer
part of the membrane and particles internalized by the
cells, the extracellular FITC was quenched with trypan
blue (TB). Interestingly, only about 20% of the FITC/PEI
incubated cells showed remaining fluorescence in flow
cytometry measurements after TB quenching, while the
corresponding number was 40% for the FITC/PEI/FA in-
cubated cells, as shown in Figure 4B. The level of fluo-
rescence for the plain FITC-particles was equal to back-
ground fluorescence, indicating that virtually no
particles were internalized in this case. This is opposite
to the results obtained by Lin et al., showing efficient
uptake of FITC-labeled mesoporous nanoparticles at
this concentration.
39
This difference can probably be as-
cribed to differences in particle size (100 nm particles
were studied by Lin et al. as compared to about 400 nm
particles in our case), as a smaller particle size may
lead to enhanced nonspecific cellular uptake. For dye-
functionalized mesoporous silica, a particle size of 50
nm has been reported to allow for the most efficient up-
take in HeLa cells.
45
Similar particle uptake was also detected after
24 h of incubation, as the fraction of FITC-positive
cells was only 1.6% for the plain particles (FITC),
whereas the corresponding values for FITC/PEI and
FITC/PEI/FA were 24% and 52%, respectively (see
Supporting Information). The data was recorded af-
ter TB quenching of extracellular fluorescence. Thus,
virtually no uptake of plain FITC-particles by the
HeLa cells was observed after 24 h. However, the
HeLa cells showed enhanced fluorescence when ex-
posed to nanoparticles functionalized with PEI, and
much more so for the FITC/PEI/FA particles, indicat-
ing a successful discrimination between the biospe-
cifically tagged and plain (FITC-labeled) particles,
Figure 8. Specific particle endocytosis of FITC/PEI/FA-functionalized silica nanoparticles. 293 cells (A) or HeLa cells (B) were left un-
treated (control) or incubated with nanoparticles (10 g/ml) for 4 h after which the extracellular fluorescence was quenched by try-
pan blue. The endocytosed particles with FITC label (green) inside CMAC-labeled (blue) cells were detected by confocal micros-
copy (A and B) or flow cytometry (C). Mean fluorescence intensity (MFI) of FITC in the cells was measured and the values were
normalized to particle endocytosis in HeLa cells (n 3; mean SEM).
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probably by recognition by the folate receptor fol-
lowed by receptor-mediated endocytosis.
Folate-Receptor Mediated Uptake. To verify that the up-
take is mediated via the folate receptor, a competition
experiment using different amounts of free folic acid in
the cell medium together with the FITC/PEI/FA
-functionalized silica nanoparticles was performed. The
number of HeLa cells that had internalized the FITC/PEI/
FA-functionalized silica particles decreased with in-
creasing concentration of free FA, as shown in Figure
5. At a free FA concentration of 3 mM the mean fluores-
cence intensity (MFI) value decreased to a value of
about 1/3 of the value measured in the absence of
free FA. The inhibition of uptake by free FA in a dose-
dependent manner demonstrates that the particle up-
take is mediated by the FA receptor. However, we note
that as some internalization of nanoparticles occurs
even at a concentration of free FA of 3 mM, some other
cellular uptake mechanisms in addition to receptor-
mediated endocytosis are operative, and our current ef-
forts focus on minimizing the influence of nonspecific
nanoparticle uptake by further optimization of the sur-
face functionality of the particles.
Cellular Toxicity. To assess cellular toxicity of the par-
ticles, we determined the number of dying cells in cul-
tures treated with high and low concentrations of FITC/
PEI/FA nanoparticles for up to 24 h as compared to
untreated cells. As the cellular uptake is fairly rapid and
can be observed already after 23 h, toxicity would
generally be observed within 24 h.
40,46
Nuclear mor-
phology as determined by confocal microscopy of DAPI
stained cells was used to assess the level of cell death
(Figure 6AB). Cytotoxicity would be clearly evident by
gross changes in nuclear morphology of cells undergo-
ing apoptosis. The results show no difference in cell
death over time in the treated versus untreated cells.
This result was further quantified by a parallel experi-
ment where we analyzed the nuclear morphology by
flow cytometry of propidium iodide (PI)-stained nuclei
(Figure 6C). The condition of the cells was furthermore
monitored by light microscopy for up to 72 h after ad-
dition of the particles with no effects observed on cell
Figure 9. Specific endocytosis of FITC/PEI/FA-functionalized silica nanoparticles in coculture of HeLa and 293 cells. The cells
were labeled with blue CMAC (HeLa) or CellTracker Red (293) and plated together overnight prior to incubation with the par-
ticles for 4 h. After incubation the extracellular fluorescence was quenched by trypan blue and the endocytosed particles
with FITC-label (green) inside blue- or red-labeled cells were detected by confocal microscopy. Scale bar 10 m. The results
are representative of two independent experiments.
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morphology (data not shown). Taken together, these
results demonstrate that there are no signs of signifi-
cant nanoparticle-induced cellular toxicity for a pro-
longed period of time.
Specific Internalization of FA-Conjugated Particles by Normal
and Tumor Originating Cells. To positively identify the abil-
ity to target the FITC/PEI/FA-functionalized silica par-
ticles to HeLa cancer cells, we compared the nanoparti-
cle uptake by HeLa and 293 cells under identical
conditions. The embryonic kidney epithelial 293 cells
express a much lower level of the folate receptor than
the HeLa cells, as shown in Figure 7, although the 293
cells do express some folate receptors (Figure 7). A
clearly higher number of the green fluorescent FITC/
PEI/FA-functionalized silica particles are detected in the
HeLa cells (Figure 8B), than in the 293 cells (Figure 8A).
Flow cytometry measurements were performed to
quantify the number of HeLa and 293 cells targeted by
FITC/PEI/FA-functionalized silica particles. The number
of HeLa cells that had internalized silica nanoparticles
was consistently 2-fold, as compared to that of 293 cells
(Supporting Information, Figure S6). Even more impor-
tantly, the mean fluorescence (MFI) values measured for
the two cell types indicated that the FITC-positive HeLa
cells had internalized 56 times more of the FITC/PEI/
FA-functionalized silica nanoparticles, than had the 293
cells, as shown in Figure 8C. The total number of par-
ticles internalized by the HeLa cancer cells was there-
fore about an order of magnitude higher than the to-
tal number of particles internalized by 293 cells, a
difference high enough to be of significant therapeuti-
cal importance. The selective uptake of FITC/PEI/FA par-
ticles by HeLa cells were additionally verified in cocul-
ture experiments, that is, where both HeLa cells and 293
cells were incubated together, as shown in Figure 9.
The fact that green emission due to FITC was observed
almost exclusively within HeLa cells confirms preferen-
tial uptake of the silica particles by the HeLa cancer cells
with elevated folate receptor expression as compared
to the noncancerous 293 cells, positively proving suc-
cessful targeting of our hybrid silica particles also un-
der coculture conditions.
CONCLUSIONS AND OUTLOOK
We have developed a selective nanoparticulate sys-
tem for cancer cell targeting based on fluorescently la-
beled, PEI-functionalized and folic acid-conjugated me-
soporous silica nanoparticles. The PEI function is grown
by surface hyperbranching polymerization, and is thus
covalently attached to the particle surface. This ensures
full stability of the PEI-layer upon any further surface
modifications and final application. Our results posi-
tively show that the PEI-mesoporous silica hybrid nano-
particles are nontoxic and furthermore can be specifi-
cally endocytosed using folic acid as the targeting
ligand even under coculture conditions. The total num-
ber of particles internalized by the folate-receptor high
cancer cells was about an order of magnitude higher
than the total number of particles internalized by folate-
receptor low normal cells, making the particles highly
promising candidates for targeted drug delivery for
cancer treatment or imaging agents for early tumor
diagnosis.
The PEI surface layer can, if desired, easily be fur-
ther modified for example by rational tuning of the par-
ticle surface charge,
37
as a complementary passive tar-
geting strategy.
47
Furthermore, covalent linking of
additional targeting ligands to the hyperbranched PEI
layer is straightforward, which could lead to further in-
creases in cell-type specificity, as tumor cells typically
overexpress multiple types of surface receptors.
48
These
aspects are currently under study in our laboratories.
METHODS
Preparation and Characterization of Mesoporous Silica Nanoparticles.
Mesoporous silica nanoparticles were prepared according to a
procedure described by Nakamura et al.,
49
with the difference
that the thiol-silane was replaced by
3-aminopropyltrimethoxysilane, APS. In a typical synthesis,
1.19 g of tetramethoxysilane (TMOS) was mixed with APS under
inert atmosphere and added to an alkaline solution containing
the structure-directing agent cetyltrimethyl ammonium chloride
(CTACl). The resulting synthesis mixture had a molar ratio of 0.9
TMOS: 0.1 APS: 1.27 CTACl: 0.26 NaOH: 1439 MeOH: 2560 H
2
O.
The sol was stirred overnight at room temperature(298 K), and
thereafter aged for8hatstatic conditions. The precipitate was
filtered off, washed with deionized water and dried at 318 K in
vacuo for 72 h. The structure-directing agent was subsequently
removed by ultrasonication in acidic (HCl) ethanol (about 0.12
w/w) three times.
50
The mesoscopic ordering of the nanoparti-
cles was confirmed by powder-XRD using a Kratky compact
small-angle system (M. Braun), particle size by dynamic light scat-
tering (Malvern ZetaSizer Nano) and scanning electron micros-
copy (Jeol JSM-6335F, Jeol Ltd., Japan); and pore size, volume,
and specific surface area by nitrogen sorption measurements
(ASAP 2010 sorptometer, Micromeritics).
Poly(ethylene imine), PEI, was grown onto the mesoporous
silica particles by hyperbranching surface polymerization accord-
ing to procedures described in our earlier publications.
8,34,35,37
Thus, before poly(ethylene imine) functionalization, the
surfactant-extracted particles were carefully vacuum-dried and
subsequently subject to argon atmosphere. The particles (0.125
g) were immersed in toluene under inert atmosphere. Catalytic
amounts of acetic acid and 45 L of aziridine were added, and
the reaction mixture was stirred overnight at 348 K. After the re-
action, the particles were filtered off, washed with copious
amounts of toluene, and vaccuum-dried for at least 24 h. The to-
tal amount PEI was calculated on the basis of thermogravimet-
ric analysis (Netzsch TGA 209).
The particles, plain and PEI-functionalized, were labeled
with FITC (fluorescein isothiocyanate) by suspending 25 mg of
particles in carbonate buffer (pH 9), to which 250 L of an etha-
nolic FITC-solution (1 mg/ml) was added and stirred for 30 min.
After this, the PEI-functionalized particles were collected by cen-
trifugation, washed with deionized water repeatedly, and subse-
quently suspended in MES buffer (pH 5). A 50 g portion of
folic acid was sonicated in MES, to which 20 Lofa1L/ml EDC-
solution (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) was
added to activate the carboxylic acid groups of FA. This solution
was rapidly added to the particle suspension, after which 25 L
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(1 mg/ml in MES) NHS (N-hydroxysuccinimide) solution was
added to the suspension. The suspension was agitated over-
night and washed with copious amounts of deionized water and
ethanol, dried in vacuo and stored at 277 K.
Electrokinetic titrations were performed between each func-
tionalization step using a Nano ZetaSizer setup (Malvern,
Worcestershire, U.K.) coupled with an MPT-2 titrator unit. The
-potential was measured as a function of pH by titrating with
0.1 or 0.5 M HCl and NaOH at 298 K.
Cell Culture and Assessment of Particle Endocytosis. HeLa cervical car-
cinoma cells and HEK 293 (Human Embryonic Kidney) cells were
cultured on 12-well plates in DMEM medium (Sigma) supple-
mented with 10% fetal calf serum (BioClear), 2 mM
L-glutamin,
100 U/ml penicillin, 100 g/ml streptomycin in 37 °C, 5% CO
2
.
FITC-, FITC/PEI- or FITC/PEI/FA-functionalized silica nanopar-
ticles were suspended in medium without antibiotics at a con-
centration of 10 g/ml. After 30 min sonication in waterbath the
medium containing the particles or control medium was ap-
plied to the 5070% confluent cells and incubated for 2, 4, or
24 h at 37 °C. For folic acid competition experiments the cells
were cultured overnight with 03 mM folic acid prior to addi-
tion of particles. The cells were trypsinized and the extracellular
fluorescence was quenched by resuspension to 200 g/ml try-
pan blue (Fluka) for 10 min at room temperature. The cells were
washed once and resuspended in PBS. The amount of endocy-
tosed particles inside cells was analyzed by LSRII or FacsCalibur
flow cytometer (FITC-A or FL-I, BD Pharmingen). The mean fluo-
rescence intensity (MFI) of the cells at FITC-A channel was mea-
sured. The data was analyzed with BD FacsDiva, and Cyflogic
softwares. GraphPad Prism software was used for the statisti-
cal analysis of the results. The bar graphs in the figures represent
mean values (SEM) from two or more independent experi-
ments as indicated in the figure legends.
For microscopical studies the cells were labeled with 20 M
CMAC CellTracker (Invitrogen) or 5 M CellTracker Red (Invitro-
gen) in medium without additives for 30 min and plated on
glass-bottom culture dishes (MatTek Corp) in normal culture me-
dium. After incubation with particles for 4 h, trypan blue was
added to culture medium (200 g/ml) and the cells were viewed
live with Zeiss LSM 510 META laser-scanning confocal micro-
scope (40 oil objective, 488 nm/405/545 nm excitation) or Le-
ica DM Ibre inverted fluorescence microscope (16,20 objec-
tive).
Assessment of Cytotoxicity. For cytotoxicity analysis HeLa cells
were plated on glass-bottom culture dishes (MatTek Corp)
and incubated with 0, 2, or 10 g/ml FITC/PEI/FA-
functionalized nanoparticles for 24 h. Cell death was deter-
mined by analyzing changes in the nuclear morphology of
DAPI-labeled cells with Zeiss LSM 510 META laser-scanning
confocal microscope (40 oil objective, 488 nm/405 excita-
tion). Alternatively the cells were collected by trypsinization
and resuspended in propidium iodide (PI) buffer (40 mM Na-
citrate, 0.3% Triton X-100, 50 g/ml PI; Sigma). After 10 min
incubation the samples were analyzed for nuclear fragmen-
tation with FacsCalibur flow cytometer (FL-3, BD Pharmin-
gen). The sub-G0/G1 peak was gated as a measure of nuclear
fragmentation.
Folate Receptor Surface Expression. Surface expression of the
Folate receptor was evaluated by indirect immunostaining us-
ing the anti-Folate receptor primary antibody (2 g/ml, clone
Mov18/ZEL, Alexis) followed by Alexa488 conjugated antimouse
secondary antibody (Alexis Biochemicals). Nonspecific fluores-
cence was assessed using the secondary antibody only. Flow cy-
tometric analyses were performed using an LSRII flow cytome-
ter (FITC-A, BD Pharmingen).
Acknowledgment. M. Ja¨rn is thanked for performing the SEM-
analysis and A. Duchanoy and B. Ufer are thanked for supplying
the schematic image. The partial financial support from the Tor,
Joe and Pentti Borg foundation as well as the financial support
from the EU project NanoEar (contract number NMP4-CT-2006-
026556) (J.M.R.) is gratefully acknowledged.
Supporting Information Available: Powder XRD pattern and ni-
trogen sorption isotherms for the studied material, as well as dy-
namic light scattering and thermogravimetry measurements.
Flow cytometry measurements of the fraction of HeLa cells that
has internalized differently functionalized nanoparticles after
24 h, as well as the fraction of HeLa as compared to 293 cells
with internalized FITC/PEI/FA-functionalized nanoparticles. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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    • "In this particular case, there are two possibilities for a successful release of the cargo from the NPs. First, NPs may remain within the endosome but the drug diffuses into the cytoplasm (Rosenholm et al., 2009). Second, NPs can escape from the endosome and be released into the cytosol. "
    Full-text · Dataset · Sep 2016 · European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences
    • "Moreover, the FA is readily internalized into cells through receptor mediated endocytosis with non-immunogenicity [12,13]. The conjugation of FA on fluorescent nanomaterials is a key strategy for developing applications as a probe in targeting and imaging cancer cells [14]. Numerous studies have been devoted to covalent conjugation of FA onto fluorescent nanomaterials, such as quantum dots (QD) [5,9] . "
    [Show abstract] [Hide abstract] ABSTRACT: An efficient approach for targeting and detecting folate-receptor (FR)-positive cancer cells was developed through non-covalent conjugation of folic acid (FA) on polyethyleneimine modified CDs (PEI-CDs). The fluorescent CDs were prepared by a facile hydrothermal method, and their surfaces were wrapped with positively charged PEI for further conjugation with FA through electrostatic interaction. The FA targeted PEI modified CDs (FA-PEI-CDs) can be used as a turn-on fluorescent nanoprobe for folate receptor (FR)-positive cancer cells in vivo and in vitro. The uptake of the designed FA-PEI-CDs by HeLa and HepG2 cancer cells was verified by confocal laser scanning microscopy after 10 min incubation and competition experiments, as well as a comparative study with FR-negative MCF-7 cells.
    Full-text · Article · Jul 2016
    • "Our nanocarrier system has already previously been comprehensively evaluated for cytotoxicity, thereby proven non-cytotoxic and biocompatible (Desai et al., 2016; Mamaeva et al., 2011; Rosenholm et al., 2009a; Senthilkumar et al., 2015; Wittig et al., 2014 ), and was thus concluded to be well suited for cellular studies. To verify that the release of cargo can be ascribed to the hydrophobic effect, i.e. the tendency of nonpolar substances to interact with each other (Chandler, 2005 ) we investigated the intracellular uptake of MSN-PEI/DiI particles and the intracellular release of DiI dye from the same particles in cancer cells, and also compared it to the uptake and dissolution behavior of a corresponding amount of free DiI compound. "
    [Show abstract] [Hide abstract] ABSTRACT: The intracellular release mechanism of hydrophobic molecules from surface-functionalized mesoporous silica nanoparticles was studied in relation to the biodegradation behavior of the nanocarrier, with the purpose of determining the dominant release mechanism for the studied drug delivery system. To be able to follow the release intracellularly real-time, a hydrophobic fluorescent dye was used as model drug. The in vitro release of the dye was investigated under varying aqueous conditions in terms of pH, polarity, protein and lipid content, presence of hydrophobic structures and ultimately, in live cancer cells. Results of investigating the drug delivery system show that the degradation and drug release mechanisms display a clear interdependency in simple aqueous solvents. In pure aqueous media, the cargo release was primarily dependent on the degradation of the nanocarrier, while in complex media, mimicking intracellular conditions, the physicochemical properties of the cargo molecule itself and its interaction with the carrier and/or surrounding media was found to be the main release-governing factors. Since the material degradation was retarded upon loading with hydrophobic guest molecules, the cargo could be efficiently delivered into live cancer cells and released intracellularly without pronounced premature release at extracellular conditions. From a rational design point of view, pinpointing the interdependency between these two processes can be of paramount importance considering future applications and fundamental understanding of the drug delivery system.
    Full-text · Article · Jun 2016
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