Biodegradable polysilsesquioxane nanoparticles as efficient contrast agents for magnetic resonance imaging

Article abstract of DOI:10.1002/smll.201300198

Polysilsesquioxane (PSQ) nanoparticles are crosslinked homopolymers formed by condensation of functionalized trialkoxysilanes, and provide an interesting platform for developing biologically and biomedically relevant nanomaterials. In this work, the design and synthesis of biodegradable PSQ particles with extremely high payloads of paramagnetic Gd(III) centers is explored, for use as efficient contrast agents for magnetic resonance imaging (MRI). Two new bis(trialkoxysilyl) derivatives of Gd(III) diethylenetriamine pentaacetate (Gd-DTPA) containing disulfide linkages are synthesized and used to form biodegradable Gd-PSQ particles by base-catalyzed condensation reactions in reverse microemulsions. The Gd-PSQ particles, PSQ-1 and PSQ-2, carry 53.8 wt% and 49.3 wt% of Gd-DTPA derivatives, respectively. In addition, the surface carboxy groups on the PSQ-2 particles can be modified with polyethylene glycol (PEG) and the anisamide (AA) ligand to enhance biocompatibility and cell uptake, respectively. The Gd-PSQ particles are readily degradable to release the constituent Gd(III) chelates in the presence of endogenous reducing agents such as cysteine and glutathione. The MR relaxivities of the Gd-PSQ particles are determined using a 3T MR scanner, with r₁ values ranging from 5.9 to 17.8 mMs^-1 on a per-Gd basis. Finally, the high sensitivity of the Gd-PSQ particles as T₁-weighted MR contrast agents is demonstrated with in vitro MR imaging of human lung and pancreatic cancer cells. The enhanced efficiency of the anisamide-functionalized PSQ-2 particles as a contrast agent is corroborated by both confocal laser scanning microscopy imaging and ICP-MS analysis of Gd content in vitro. The applicability of polysilsesquioxane (PSQ) nanoparticles with cleavable Gd chelates as T₁-weighted MRI contrast agents is evaluated in vitro. The chelates are covalently linked via a redox-responsive disulfide moiety, and the platform is further functionalized to improve biocompatibility and cell uptake. The Gd-PSQ particles show high r ₁ relaxivity values and strong enhancement of T₁-weighted images of human lung and pancreatic cancer cells in vitro. Copyright

Full text of DOI:10.1002/smll.201300198

Contrast Agents
Biodegradable Polysilsesquioxane Nanoparticles as
Efficient Contrast Agents for Magnetic Resonance Imaging
Juan L. Vivero-Escoto, William J. Rieter, Honam Lau, Rachel C. Huxford-Phillips,
and Wenbin Lin*
P
olysilsesquioxane (PSQ) nanoparticles are crosslinked homopolymers formed
by condensation of functionalized trialkoxysilanes, and provide an interesting
platform for developing biologically and biomedically relevant nanomaterials. In
this work, the design and synthesis of biodegradable PSQ particles with extremely
high payloads of paramagnetic Gd(III) centers is explored, for use as efficient
contrast agents for magnetic resonance imaging (MRI). Two new bis(trialkoxysilyl)
derivatives of Gd(III) diethylenetriamine pentaacetate (Gd-DTPA) containing
disulfide linkages are synthesized and used to form biodegradable Gd-PSQ particles
by base-catalyzed condensation reactions in reverse microemulsions. The Gd-PSQ
particles, PSQ-1 and PSQ-2, carry 53.8 wt% and 49.3 wt% of Gd-DTPA derivatives,
respectively. In addition, the surface carboxy groups on the PSQ-2 particles can be
modified with polyethylene glycol (PEG) and the anisamide (AA) ligand to enhance
biocompatibility and cell uptake, respectively. The Gd-PSQ particles are readily
degradable to release the constituent Gd(III) chelates in the presence of endogenous
reducing agents such as cysteine and glutathione. The MR relaxivities of the Gd-PSQ
particles are determined using a 3T MR scanner, with r values ranging from 5.9 to
1
−
1
1
7.8 mMs on a per-Gd basis. Finally, the high sensitivity of the Gd-PSQ particles
as T -weighted MR contrast agents is demonstrated with in vitro MR imaging of
1
human lung and pancreatic cancer cells. The enhanced efficiency of the anisamide-
functionalized PSQ-2 particles as a contrast agent is corroborated by both confocal
laser scanning microscopy imaging and ICP-MS analysis of Gd content in vitro.
increased relative surface area, as well as the quantum con-
1
. Introduction
finement effect in some systems. In addition, these particu-
late materials can be functionalized to endow key properties
for in vivo applications such as biocompatibility, long blood
circulation time, target-specificity, and programmed clear-
ance mechanisms. Such nanoprobes are beneficial to various
imaging modalities, in particular, magnetic resonance imaging
(MRI).
MRI is one of the most powerful noninvasive biomed-
ical imaging technologies. However, MRI is also among the
least sensitive imaging techniques and often requires the
aid of contrast agents in order to produce adequate con-
trasts between normal and diseased tissues. Currently used
MRI contrast agents are either macrocyclic gadolinium(III)
chelates or simple manganese(II) molecules that lack
Nanoparticles are emerging as powerful contrast agents for
medical imaging and biological diagnostics. The outstanding
properties of such nanoscale materials are attributed to the
[
1]
Dr. J. L. Vivero-Escoto,[+] Dr. W. J. Rieter, H. Lau,
R. C. Huxford-Phillips, Prof. W. Lin
Department of Chemistry
University of North Carolina
Chapel Hill, North Carolina 27599, USA
E-mail: wlin@email.unc.edu
[+]Present Address: Department of Chemistry, Burson 200, University of
North Carolina at Charlotte, Charlotte, NC 28223, USA
DOI: 10.1002/smll.201300198
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J. L. Vivero-Escoto et al.
than surface-functionalized silica-based
materials. In addition, the physicochem-
ical properties of PSQ nanoparticles can
be more easily tuned by changing the
monomer properties than in silica-based
systems. We wish to take advantage of
these attractive properties to formulate
PSQ-based MRI contrast agents with high
payloads of Gd chelates, programmed
clearance mechanisms, and enhanced MR
imaging properties. Herein, we report the
Scheme 1. Synthesis of Gd-PSQ nanoparticles in reverse microemulsions. Two different
Gd-chelates (1 and 2) were used to afford PSQ-1 and PSQ-2 particles with high r1 MR
relaxivities. PSQ-2 was further functionalized with PEG or anisamide-PEG via the surface synthesis and characterization of Gd-PSQ
carboxy groups.
nanoparticles and their application as T1-
weighted MR contrast agents (Scheme 1).
sensitivity.[2] In addition, these small molecule contrast agents These particles contain Gd(III) chelates covalently linked via
tend to have unfavorable pharmacokinetics. As a result, they a labile disulfide bond, which bestow redox-responsive degra-
are typically administered in high doses in order to achieve dable properties to this system. The Gd-PSQ nanoparticles
adequate contrasts. Nanoparticle-based MRI contrast agents were post-synthetically modified with polyethylene glycol
can potentially overcome this limitation by providing more (PEG) chains and the anisamide (AA) ligand to enhance
efficient signal enhancement as a result of the enhanced relax- biocompatibility and cell uptake, respectively. The effective-
ivity on a per magnetic center basis as well as their ability to ness of the PSQ particles as efficient optical imaging and
carry extremely large payloads of active magnetic centers. MRI contrast agents was successfully demonstrated using
Moreover, these MRI nanoprobes can be further modified human lung (H460) and pancreatic (AsPC-1) cancer cells.
to improve biocompatibility, long blood circulation time, and
target-specificity. Several platforms have been used to develop
nanoparticle-based MRI contrast agents, including dendrimers,
[3]
2. Results and Discussion
polymers, liposomes, and hybrid or inorganic nanoparticles.
We and others have reported the development of
mesoporous silica nanoparticle (MSN) based MRI contrast 2.1. Synthesis and Characterization of Gd-PSQ Nanoparticles
[
4]
agents. We have demonstrated that this platform has an
extraordinary ability to enhance MR images both in vitro Two new Gd(III) diethylenetriamine pentaacetate (Gd-
[4d,e,g]
and in vivo.
However, significant amounts of silica DTPA) derivatives (Scheme 1) were used as the monomers
matrices are used to carry the Gd chelates in the MSN sys- for the synthesis of the Gd-PSQ MRI contrast agents. Gd(III)
tems. Moreover, many of these nanoprobes cannot be quickly [bis(triethoxysilylpropyl cystamide) diethylenetriamine pen-
excreted after MRI examination, leading to potential tissue taacetic acid] (1), was synthesized in a multistep sequence
[
5]
accumulation of toxic Gd(III) ions. This slower clearance (Scheme S1, SI). 2-(pyridin-2-yldisulfanyl)cysteamine (a)
of Gd(III) chelates has been associated to a fatal condition was first prepared by treating cysteamine and 2,2′-dipyridyl
[
6]
known as nephrogenic systemic fibrosis (NSF). For these disulfide in a methanolic solution containing acetic acid,
reasons, it is imperative to develop new nanoparticulate plat- followed by treatment with one half equivalent of DTPA
forms that not only carry high payloads of Gd chelates but bisanhydride in anhydrous pyridine to afford bis-cystamide-
also can be readily eliminated from the body after carrying diethylenetriamine pentaacetic acid (b). The corresponding
out their diagnostic functions.
Gd(III) complex c was formed by reacting one equivalent of
Polysilsesquioxane (PSQ) nanoparticles are crosslinked GdCl with the deprotonated form of b. Finally, the desired
3
homopolymers formed by condensation of functionalized Gd-chelate (1) was produced by reacting two equivalents
trialkoxysilanes or bis(trialkoxysilanes). PSQ materials have of 3-mercaptopropyltriethoxysilane with c in dry methanol.
recently attracted a great deal of attention as a class of versa- Gd(III) [bis(triethoxysilylpropyl cysteinamido) diethylen-
tile functional hybrid particles with tunable size, morphology, etriamine pentaacetic acid] (2) was synthesized in a similar
and chemical properties.[7] We believe that PSQ particles fashion with cysteine hydrochloride in place of cysteamine
provide an interesting platform for developing biologi- hydrochloride in the reaction sequence (Scheme S1, SI). The
cally and biomedically relevant nanomaterials. We recently disulfide linkages in both Gd complexes 1 and 2 can be rap-
demonstrated the design of PSQ nanoparticles containing idly reduced by endogenous reducing agents such as cysteine
a high loading of a chemotherapeutic agent (oxaliplatin) as and glutathione to endow biodegradability to the resulting
[
8]
an excellent delivery vehicle for cancer therapeutics. PSQ PSQ particles (see Section 2.3). In addition, the Gd complex
particles not only retain most of the attractive features of 2 contains orthogonal carboxylic acid functionalities, which
silica-based materials for biomedical applications such as can be used to post-synthetically functionalize the resulting
well-defined and tunable structures, surface chemistry, and PSQ nanoparticles as described below.
biocompatibility but also offer additional advantages. For
example, the PSQ platform is essentially carrier free, allowing base-catalyzed condensation reactions in
much higher loadings of therapeutic and/or imaging agents and a ternary reverse microemulsion system, respectively.
PSQ particles based on 1 and 2 were synthesized by
quaternary
a
3
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Biodegradable Polysilsesquioxane Nanoparticles for MRI
Figure 1. Characterization of PSQ nanoparticles. a) SEM image of PSQ- Figure 2. Characterization of PSQ nanoparticles. a) SEM image of PSQ-
1
; b) DLS plot of PSQ-1 in PBS; c) TGA profile for PSQ-1 (black), and 2; b) DLS plots of PSQ-2 (square), PEG-PSQ-2 (circle), and AA-PEG-PSQ-2
PSQ-2 (gray).
(triangle) in PBS; c) TGA profiles for PSQ-2 (black), PEG-PSQ-2 (gray),
and AA-PEG-PSQ-2 (light gray).
The resulting PSQ-1 and PSQ-2 nanoparticles were pre- discrepancy between the SEM and DLS results is probably
cipitated by the addition of ethanol, followed by repeated due to a combination of the slight aggregation of PSQ-1 in
[7c]
washes with ethanol. The particle sizes and morphologies PBS and its hydrogel-like behavior. SEM images of PSQ-
were characterized by scanning electron microscopy (SEM) 2 showed fairly spherical nanoparticles of 100–150 nm in
and dynamic light scattering (DLS). As shown in Figure 1a , diameter (Figure 2a). The DLS measurements of PSQ-2 gave
SEM images of PSQ-1 showed aggregated nanoparticles of a hydrodynamic diameter of 220 nm (PDI = 0.062) in PBS
roughly 30 nm in diameter. However, DLS measurements on (Table 1 and Figure 2b). PSQ-1 and PSQ-2 exhibited negative
the same PSQ-1 gave a hydrodynamic diameter (Z-average) surface charges (ζ potentials) of −15.0 and −22.4 mV, respec-
of 123 nm in a phosphate buffered saline solution (PBS, 1 tively, in PBS buffer. Thermogravimetric analysis (TGA) and
mM) (polydispersity index, PDI = 0.15) (Figure 1b). The inductively-coupled plasma mass spectrometry (ICP-MS)
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J. L. Vivero-Escoto et al.
T a b l e 1 . Detailed characterization of Gd-PSQ nanoparticles.
Material
Z-average
PDIa)
ζ-potential Gd content PEG content
a)
a)
[
nm]
[mV]
[wt%]
[wt%]
PSQ-1
123.0 ± 2.6 0.15 ± 0.04 −15.0 ± 3.6 14.3 ± 1.4
219.5 ± 1.4 0.06 ± 0.02 −22.4 ± 2.2 15.7 ± 3.2
200.4 ± 3.6 0.09 ± 0.03 −1.9 ± 2.1 12.3 ± 4.5
—
PSQ-2
—
PEG-PSQ-2
6.3
7.4
AA-PEG-PSQ-2 292.5 ± 3.8 0.25 ± 0.01 −3.1 ± 2.3 10.9 ± 5.8
a)Measured in PBS (1 mM, pH 7.4).
were used to determine the loadings of Gd chelates in the
PSQ particles. PSQ-1 showed a 44.2 wt% weight loss for
the organic component (Figure 1c). ICP-MS measurements
of PSQ-1 gave a Gd loading of 14.3 wt%. PSQ-2 showed a
41.3 wt% organic weight loss by TGA and 15.7 wt% Gd by
ICP-MS (Table 1 and Figure 2c). Interestingly, owing to the
lack of carriers, Gd-PSQ particles have three times higher
loadings of paramagnetic centers than those of mesoporous
[4e,g]
silica nanoparticles.
The surface carboxy groups on PSQ-2 allow their post-
synthetic functionalization to increase biocompatibility and
cell uptake. Polyethylene glycol (PEG) is the preferred method
of increasing biocompatibility and imparting stealth properties
Figure 3. T - (a) and T -weighted (b) MR phantom images of PSQ-2,
PEG-PSQ-2, and AA-PEG-PSQ-2 at various Gd concentrations.
1
2
[9]
to nanoparticles. PSQ-2 particles were PEGylated through
a coupling reaction between the carboxylic acid groups on
the surface of the nanoparticles and an amino-functionalized compare favorably to other nanoparticle-based MRI contrast
PEG(5K) using 1-ethyl-3-(3-dimethylaminopropyl) carbodi- agents and are superior to the small molecule contrast agent
−
1 −1
imide (EDC) as a coupling agent to afford PEG-PSQ-2. SEM Magnevist, which has an r value of 4.9 mM s under
1
[
3f]
images did not show any appreciable change in the morphology similar conditions. The enhanced r relaxivities for nano-
1
or size of the PEG-PSQ-2 nanoparticles (see Figure S1, SI). particles can in part be attributed to their slower tumbling
[
11]
The DLS measurements showed that PEG-PSQ-2 particles rates.
have a similar hydrodynamic diameter as that of PSQ-2 (Figure fully internalized by monocyte cells as shown in the T - and
Interestingly, PSQ-1 nanoparticles can be success-
1
2
b and Table 1). However, the surface charge of the mate- T -weighted MRI images of cell pellets (see Figure S3, SI).
2
rial measured by the ζ potential increased dramatically from
22.4 mV to −1.9 mV, indicating the presence of a PEG layer PSQ-2 nanoparticles were also determined on a 3T MR
The MR relaxivities of the PEG-PSQ-2 and AA-PEG-
−
−
1 −1
that shields the negative surface charge of PSQ-2. The exist- scanner. On a per Gd basis, the r values were 8.4 mM s
ence of PEG was further confirmed by TGA; an increase in and 5.9 mM s and the r values were 33.0 mM s and
weight loss of 6.3% was observed after PEGylation (Figure 2c 7.0 mM s for PEG-PSQ-2 and AA-PEG-PSQ-2, respec-
1
−1 −1
−1 −1
2
−1 −1
and Table 1). In a similar way, the AA-terminated PEG was tively (Figure S2c,d, SI). The r relaxivity values for the modi-
1
attached to the surface of PSQ-2 using a mixture of NH -PEG fied PSQ particles decreased in comparison to that of PSQ-2,
2
and amino-functionalized AA-PEG (7:3 wt/wt) to afford AA- presumably due to the presence of PEG and AA-PEG chains
PEG-PSQ-2 particles. The AA moiety is an effective ligand for that inhibit the interactions of the Gd centers with sur-
[
12]
sigma receptors, which are overexpressed in a variety of human rounding water molecules.
[10]
and rodent cancer cell lines.
After functionalization with
AA-PEG, the nanoparticles exhibited an increase of 70 nm in
their hydrodynamic diameter (Z-average = 292.5 nm) and a ζ
potential of −3.1 mV (Figure 2b and Table 1). TGA showed an
increase of organic weight loss of 7.4%.
2
.3. Biodegradability of Gd-PSQ Particles under Reducing
Environments
The Gd-PSQ particles should be stable under normal physi-
ological conditions, but can readily degrade to release the Gd
chelates as a result of the reductive cleavage of the disulfide
bonds by endogenous biomolecules, such as glutathione and
2.2. Determination of MR Relaxivities of Gd-PSQ Particles
The relaxivities for the Gd-PSQ particles were measured cysteine. The Gd chelate monomers could be cleared via
with a 3T MR scanner (Figure 3). On a per Gd basis, PSQ-1 the renal excretion pathway in vivo to reduce the potential
−
1 −1
[4g,13]
and PSQ-2 have r values of 17.8 and 13.1 mM s , respec- toxicity of the Gd-PSQ particles.
Release experiments
1
−1 −1
tively, and r values of 19.5 and 28.6 mM s , respectively revealed that both PSQ-1 and PSQ-2 particles are stable in the
2
(
Figure S2a,b, SI). The r relaxivities of the Gd-PSQ particles absence of reducing agents, with only 5% or less background
1
3
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Biodegradable Polysilsesquioxane Nanoparticles for MRI
Figure 5. T -weighted images of H460 and AsPC-1 cell pellets. a) The
1
images from left to right correspond to H460 cells incubated with no
particles, AA-PEG-PSQ-2 (100 μg/mL), PEG-PSQ-2 (100 μg/mL), and
PSQ-2 (100 μg/mL). b) The images from left to right correspond to
AsPC-1 cells incubated with no particles, AA-PEG-PSQ-2 (100 μg/mL),
PEG-PSQ-2 (100 μg/mL), and PSQ-2 (100 μg/mL). Scale bar is 5 mm.
Figure 4. Release profiles of Gd chelates from PSQ-1 (squares), PSQ-2
(circles), and PEG-PSQ-2 (triangles) in the presence of 10 mM cysteine
at 37 °C.
release over a few hours (Figure 4). However, after the addi-
tion of 10 mM cysteine, Gd chelates were quickly released shown in Figure 5. Significant positive signal enhancements
from the PSQ-1 and PSQ-2 particles with half-lives (t1/2) of were observed in the T -weighted MR images of both H460
1
5
h and 12 h, respectively. In both particles, more than 90% and AsPC-1 cell pellets incubated with all versions of PSQ-
of the Gd chelates were released after two days of incubation 2 particles when compared to cell pellets that were not
with cysteine. In the case of PSQ-2, the steric effect around incubated with nanoparticles. Moreover, the T -weighted
1
the disulfide bonds significantly affected the particle degrada- images of the H460 and AsPC-1 cell pellets incubated with
[14]
tion rate in the presence of reducing agents. After PEGyla- the AA-PEG-PSQ-2 particles showed much higher signal
tion, the t1/2 of Gd chelate release further increased to 18 h, enhancements than those incubated with non-modified PSQ-
consistent with the additional steric hindrance from the PEG 2 particles. For each of the cell lines, the T -weighted MR
1
polymer (Figure 4). The ability to tune the degradation rates signal intensity appears to follow the increasing trend of
of Gd-PSQ particles via steric control over the disulfide bond no particle < PEG-PSQ-2 < PSQ-2 < AA-PEG-PSQ-2. This
should prove important for optimizing in vivo performance behavior was confirmed by quantitative ICP-MS measure-
of this nanoparticulate MR contrast agent platform in the ments of the amounts of Gd ions internalized by cancer cells
future.
(Figure 6). The ICP-MS data also showed that the internali-
zation of PEG-PSQ-2 particles was inhibited compared to
PSQ-2 (Figure 6); however, the presence of the AA targeting
group enhanced the amount of Gd internalized by the cancer
cells. It has been shown that coating nanoparticles with PEG
decreases the non-specific binding to the cell membrane, thus
2.4. In Vitro Performance of Gd-PSQ Particles as Efficient MRI
and Optical Imaging Contrast Agents
[15]
T -weighted MR images of human lung H460 and pancreatic reducing the nonspecific accumulation in cells.
One the
1
AsPC-1 cancer cells after incubation with PSQ-2 particles are other hand, sigma receptors (e.g., sigma-2) are more highly
Figure 6. Amount of Gd(III) ions internalized by H460 (left) and AsPC-1 (right) cells incubated with PSQ materials for 12 h with 100 μg mL−1
.
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J. L. Vivero-Escoto et al.
3
. Conclusion
We have synthesized and characterized
Gd-PSQ nanoparticles with a high loading
of magnetic centers and a strong ability to
enhance MRI contrast. The Gd-PSQ par-
ticles were constructed from two different
Gd-chelates containing redox-responsive
disulfide bonds, and were biodegradable in
the presence of an endogenous reducing
agent. The Gd-PSQ particles were further
functionalized with PEG and anisamide-
PEG chains to enhance biocompatibility
and cell uptake, respectively. The effective-
ness of Gd-PSQ particles as T -weighted
1
MRI contrast agents was demonstrated in
vitro on a 3T MR scanner using H460 and
AsPC-1 cancer cells, which was confirmed
by confocal laser scanning microscopy and
ICP-MS studies. The high loading of Gd
chelates, biodegradability, and high MR
sensitivity of Gd-PSQ particles make them
a promising new class of efficient MRI
contrast agents.
4. Experimental Section
Synthesis of 2-(Pyridin-2-yldisulfanyl)
Figure 7. Confocal microscope images of H460 cancer cells incubated with a) FITC-labeled
PSQ-2; b) FITC-labeled PEG-PSQ-2; and c) FITC-labeled AA-PEG-PSQ-2. The figures from left to
right depict bright field images, green fluorescence of FITC-labeled PSQ materials, and merged
images of bright field and green fluorescence with DRAQ5-stained nuclei, respectively. The procedure.
cysteamine hydrochloride (a) : The synthesis of
a was carried out by an established literature
[16]
cells were incubated in the presence of Gd-PSQ particles for 12 h at a concentration of
00 μg/mL. Scale bar is 20 μm.
Synthesis of Bis(2-pyridyldisulfanyl eth-
ylamido)diethylenetriamine pentaacetic acid
dihydrochloride (b) : A mixture of a (0.80 g,
1
expressed in cancer cells compared to normal cells, leading to 3.60 mmol) and DTPA dianhydride (0.52 g, 1.44 mmol) in anhy-
[
10a,b]
enhanced cellular uptake of AA-PEG-PSQ-2.
drous pyridine (10 mL) was magnetically stirred for 24 h. The crude
The enhanced efficacy of AA-PEG-PSQ-2 particles as product was isolated by precipitation with diethyl ether, and puri-
MRI contrast agents was further confirmed by confocal fied by repeated dissolution (up to 4 times) in a minimal volume
laser scanning microscopic (CLSM) imaging of H460 and of methanol followed by precipitation with diethyl ether to yield
1
AsPC-1 cells. The AA-PEG-PSQ-2, PEG-PSQ-2, and PSQ-2 an off-white solid of b in a quantitative yield. H NMR (300 MHz,
nanoparticles were labeled with fluorescein isothiocyanate D2O, ppm): 2.97 (t, 4H), 3.30 (s, 8H), 3.52 (t, 4H), 3.69–3.78
(
FITC) to enable optical imaging of the particles (Figure 7 (10H), 7.52 (t, 2H), 8.00 (d, 2H), 8.10 (t, 2H), 8.45 (d, 2H). MS (ESI
and Figure 8). As shown in the confocal micrographs, the positive ion): m/Z = 765.1 (expected 765.15), 730.1 (expected
+
+
AA-PEG-PSQ-2 nanoparticles (Figures 7c,8c) were internal- 730.18), and 801.1 (expected 801.11) for [M-Cl+H] , [M-2Cl+H] ,
+
ized by both cancer cell lines in a higher magnitude than the and [M+H] , respectively.
non-targeted materials (Figures 7a,8a and 7b,8b). In addition,
Synthesis of Gd(III) [Bis(2-pyridyldisulfanyl ethylamido)diethyl-
competitive binding assay experiments with the anisamide enetriamine pentaacetic acid] (c): The pH of a solution of b (0.73 g,
ligand confirmed the enhanced uptake of AA-PEG-PSQ-2. 1.00 mmol) in water (10 mL) was adjusted from ∼2 to ∼10 via the
ICP-MS analyses indicated that pre-incubation of both H-460 addition of dilute NaOH (4.0 mL of 1 M, 4.0 eq). To this solution,
and AsPC-1 cells with PEG-anisamide (1, 5, and 10 mM) GdCl (0.364 g, 0.98 mmol) in water (1 mL) was added dropwise.
3
significantly reduced the uptake of AA-PEG-PSQ-2 but not The resulting solution with a pH value of ∼3.5 was magnetically
PEG-PSQ-2 when compared to those without pre-incuba- stirred for an additional 6 h. The solvent was removed via rotary
tion with the anisamide ligand (Figure S4, SI). These results evaporation. The crude product was purified by repeated dissolu-
not only demonstrate the utility of the Gd-PSQ particles as tion (up to 4 times) in a minimal volume of methanol followed by
efficient T -weighted MRI contrast agents but also point to precipitation with diethyl ether to lead to an off-white solid of c in
1
the potential of detecting cancer cells by MRI and optical a quantitative yield. MS (ESI positive ion): m/Z = 885.1 (expected
imaging in a target-specific fashion via conjugation of tar- 885.11), 906.9 (expected 906.10), and 956.0 (expected 956.01)
+
+
+
geting ligands to the surfaces of Gd-PSQ particles.
for [M-2Cl+2H] , [M-2Cl+Na+H] , and [M+2H] , respectively.
3
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Biodegradable Polysilsesquioxane Nanoparticles for MRI
+
+
(
expected 460.95), for [M+H] , and [2M+H] ,
respectively.
Synthesis of Bis(2-pyridin-2-yldisulfanyl
cysteineamido)diethylenetriamine pentaacetic
acid dihydrochloride (b’) : A solution of a’
(1.95 g, 7.06 mmol) in 45 mL of dry DMSO
was added dropwise to a solution of DTPA
dianhydride (1.26 g; 43.53 mmol) in 45 mL
of dry DMSO. After the addition of diisopropy-
lethylamine (DIEA) (1.23 mL, 7.06 mmol), the
resulting solution was stirred at room temper-
ature for 48 h. The product was precipitated
by the addition of acetone and diethyl ether.
The solid residue was purified by repeated
dissolution (up to 4 times) in a minimal
volume of methanol followed by precipita-
tion with diethyl ether. Yield: 1.50 g (52%). 1H
NMR (400 MHz, DMSO-d , ppm): 2.92 (t, 4H),
6
3
(
7
(
.08 (t, 4H), 3.29 (d, 4H), 3.34 (s, 4H), 3.43
s, 4H), 3.57 (s, 2H), 4.48 (t, 2H), 7.22 (t, 2H),
.74 (t, 2H), 7.79 (t, 2H), 8.44 (d, 2H), 8.49
s, 1H-NH), 8.51 (s, 1H-NH). MS (ESI posi-
tive ion): m/Z = 818.16 (expected 818.15),
+
8
40.14 (expected 840.14), for [M+H] , and
+
[M+Na] , respectively.
Synthesis of Gd(III) [Bis(2-pyridyldisul-
fanyl cysteinamido)diethylenetriamine pen-
taacetic acid] (c’). The pH of a solution of b’
Figure 8. Confocal microscope images of AsPC-1 cancer cells incubated with a) FITC-labeled (1.00 g, 1.22 mmol) in water (14 mL) was
PSQ-2, b) FITC-labeled PEG-PSQ-2, and c) FITC-labeled AA-PEG-PSQ-2. The figures from left to
adjusted from ∼3.5 to ∼9.3 via the addition
right depict bright field images, green fluorescence of FITC-labeled PSQ materials, and merged
of dilute NaOH (∼7.1 mL, 1 M, 5.8 eq). To this
images of bright field and green fluorescence with DRAQ5-stained nuclei, respectively. The
solution GdCl (0.43 g, 1.16 mmol, 0.95 eq)
3
cells were incubated in the presence of Gd-PSQ particles for 12 h at a concentration of
in water (2 mL) was added dropwise. The
resulting solution with a pH of ∼4.5 was mag-
100 μg/mL. Scale bar is 20 μm.
netically stirred for an additional 6 h at room
Synthesis of Gd(III) [Bis(triethoxysilylpropyl cysteamido)dieth- temperature. The solvent was removed via rotary evaporation. The
ylenetriamine pentaacetic acid] (1) : To a solution of c (0.20 g, solid residue was purified by repeated dissolution (up to 5 times)
.22 mmol) in ∼3.5 mL of methanol 3-mercaptopropyl triethoxysi- in a minimal volume of methanol followed by precipitation with
0
lane (0.13 mg, 0.56 mmol) was added, and the resulting solution diethyl ether to yield an off-white product. Yield: 1.03 g (95%).
was magnetically stirred at room temperature for ∼12 h. Diethyl MS (ESI positive ion): m/Z = 995.04 (expected 995.05), 1017.01
+
ether was added to the solution to precipitate the product, which (expected 1017.03), and 1039.00 (expected 1039.03) for [M+Na] ,
+
+
was further purified by repeated dissolution (up to 4 times) in a [M-H+2Na] , and [M-2H+3Na] , respectively.
minimal volume of methanol followed by precipitation with diethyl
Synthesis of Gd(III) [Bis(triethoxysilylpropyl cysteinamido)
ether to give an off-white solid of 1. Yield: 185 mg (73%). MS (ESI diethylenetriamine pentaacetic acid] (2) : To a solution of c’
positive ion): m/Z = 869.3 (expected 869.84), [M-6EtO+2H]+.
(0.80 g, 0.81 mmol) in 67 mL of methanol 3-mercaptopropyl tri-
Synthesis of 2-(Pyridin-2-yldisulfanyl)cysteine hydrochloride ethoxysilane (454 μL, 1.78 mmol) was added. The resulting solu-
(
a’) :[14] A solution of cysteine hydrochloride (1.58 g, 10.0 mmol) tion was stirred at room temperature for 24 h. After the reaction
in methanol (20 mL) was added dropwise to a magnetically was complete, diethyl ether was added to the solution to precipi-
stirred mixture of 2,2′-dipyridyl disulfide (4.4062 g, 20.0 mmol) tate the product, which was then purified by repeated dissolution
and acetic acid (800 μL) in methanol (20 mL) over a period of (up to 4 times) in a minimal volume of methanol followed by pre-
3
0 min. After stirring at room temperature for an additional 24 h, cipitation with diethyl ether to afford an off-white product. Yield:
the solvent was removed from the mixture by rotary evaporation. 0.65 mg (65.6%). MS (ESI positive ion): m/Z = 955.8 (expected
+
The product was isolated by precipitating from a concentrated 955.82), [M-6EtO] .
methanol solution of the crude mixture with diethyl ether. This
Synthesis of PSQ-1: A solution of 1 (57.0 mg, 0.05 mmol)
washing/precipitation procedure was repeated 5 times to lead to a in water (7.6 mL) was freshly prepared and added to a mag-
1
white crystalline product. Yield: 2.14 g (80.0%). H NMR (400 MHz, netically stirred solution of 0.3 M Triton X-100/1.5 M 1-hexanol/
DMSO-d , ppm): 3.39 (m, 2H), 4.19 (t, 1H), 7.31 (t, 1H), 7.735 (d, cyclohexane (100 mL), quickly followed by the addition of aqueous
6
1
H) 7.82 (t, 1H), 8.52 (d, 1H), 8.78 (s, broad 2H corresponding to NH OH (0.50 mL). The resulting microemulsion with a W value of
4
NH ). MS (ESI positive ion): m/Z = 230.8 (expected 230.85), 460.9 15 was stirred at room temperature for an additional 24 h. The
2
small 2013, 9, No. 20, 3523–3531
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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full papers
J. L. Vivero-Escoto et al.
PSQ-1 particles were isolated by the addition of an equivalent with additional dry DMSO to make a 6 mM FITC-AP-TES solution.
volume of ethanol, followed by centrifugation at 13 000 rpm. The The FITC-PSQ-2 nanoparticles were prepared in a similar fashion as
particles were subsequently washed with ethanol before being PSQ-2 with the exception of adding 50 μL of the FITC-AP-TES solu-
redispersed in ethanol. Low molecular weight contaminants were tion to the reverse microemulsion. FITC-PSQ-2 particles were modi-
removed by dialyzing the material against dilute phosphate buff- fied with PEG and AA-PEG in the same fashion as PSQ-2.
ered saline (pH 7) using 3500 MW cutoff cellulose dialysis tubing.
Confocal Microscopy Imaging of H460 and AsPC-1 Cells: Either
The remaining nanoparticles were collected and dispersed in dis- H460 or AsPC-1 cells were plated in a 6-well plate with silanized
5
tilled water. Yield: 23.9 mg (42.0%).
coverslips at a cell density of 3 × 10 cells/well in 1 mL media per
Synthesis of PSQ-2: A solution of 2 (23.4 mg, 0.019 mmol) well. The plate was incubated for 24 h to promote cell attachment
in water (2.03 mL) was freshly prepared and added to a magneti- (5% CO , 37 °C). After the media was removed, wells were washed
2
cally stirred solution of 0.3 M Triton X-100/cyclohexane (37.5 mL), once with PBS. Fresh media containing FITC-labeled PEG-PSQ-2 or
−
1
quickly followed by the addition of aqueous NH OH (0.19 mL). AA-PEG-PSQ-2; was then added (100 μg mL ). The plates were
4
The resulting microemulsion was stirred at room temperature for incubated for 12 h. After the media was removed, wells washed
an additional 24 h. The PSQ-2 particles were isolated by the addi- with PBS and 1 mL fresh media added to each well. Coverslips
tion of an equivalent volume of ethanol followed by centrifugation were adhered onto glass slides with antifade mounting medium.
at 13 000 rpm. The particles were subsequently washed with eth- Images were obtained using a Zeiss LSM5 Pascal inverted confocal
anol, PBS (pH 3.0, 10 mM), and PBS (pH 7.4, 10 mM) before being laser scanning microscope. The FITC dye was imaged using 490 nm
redispersed in ethanol. Low molecular weight contaminants were excitation and a 525 nm long pass emission filter.
removed by dialysis of the material against dilute PBS (pH 7.4,
T1-Weighted MR Imaging of Monocyte Cells: Monocyte cells
1
8
mM) using 3500 MW cutoff cellulose dialysis tubing. Yield: were trypsinized for 5 min at 37 °C and 5% CO before collection by
2
.5 mg (36.4%).
low speed centrifugation. The cell concentration was determined
6
Release Profile Studies: 4.0 mL of an ethanolic suspension of by trypan blue exclusion. Approximately 2.0 × 10 monocytes were
PSQ particles (0.86 mg/mL) were centrifuged to isolate the parti- placed in a culture dish with 1 mL of media and 200 μL of PSQ-1
cles, which were then redispersed in 5 mL of PBS (1 mM, pH 7.4). nanoparticles (0.100 mg). After 1 h of incubation, the cells were
This suspension was placed inside a piece of dialysis tubing washed with fresh media twice and pelleted. A final layer of PBS
(
3500 MW cutoff). 400 mL of PBS (1 mM, pH 7.4) in an air-tight (200 μL) was added on top of the pellets for MR imaging studies.
container and was preheated under nitrogen at 37 °C in an oil T1-Weighted MR Imaging of H460 and AsPC-1 Cells: Confluent
bath. The dialysis bag containing the particles was then quickly H460 or AsPC-1 cells were trypsinized, and an aliquot of cell suspen-
submerged in the PBS buffer. Aliquots were removed at various sion was added to each 25 mL culture flask to obtain a cell density of
6
time points to establish the baseline release. After a few hours, a 1.0 × 10 cells/flask, followed by 7 mL RPMI-1640 complete growth
5
0 mL aliquot was removed from the container, and replaced with medium (Cellgro). The flasks were incubated (37 °C, 5% CO ) for
2
a 50 mL solution containing 0.63 g (4.0 mmol) of L-cysteine·HCl to 36 h. The media was then removed and replaced with 5.0 mL fresh
give a final cysteine concentration of 10 mM. Aliquots were again media containing PSQ-2, PEG-PSQ-2, and AA-PEG-PSQ-2. The final
−1
removed at various time points to establish the cysteine-triggered Gd-PSQ concentration was 100 μg mL . After incubating for 12 h,
release profiles. For the ICP-MS analysis, each aliquot removed the cells were trypsinized, resuspended in 200 μL PBS, and cen-
from the container was diluted in 3.0 mL of 0.6 M nitric acid. The trifuged at 3000 rpm for 15 min to obtain cell pellets. T -weighted
1
Gd content of each sample was then measured using ICP-MS.
images of the pellets were obtained on a 3.0 T MR scanner.
PEGylation of PSQ-2: The carboxylic acid groups in PSQ-2 were
Determination of the Amount of Gd Internalized by H460 and
used to couple with amino-polyethyleneglycol monomethyl ether AsPC-1 Cells: The cell pellets were prepared in the same fashion
H2N-PEG(5K)] via an amide linkage. PSQ-2 (10 mg) was treated as described above for the MR imaging experiment. Cell pellets
with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC, 50 mg) were digested in concentrated HNO for 12 h and diluted to 4 mL
[
3
(
coupling agent) and H N-PEG(5K) (100 mg) in dry acetonitrile at with MilliQ water. The samples were filtered through Nalgene PTFE
2
room temperature for 24 h to afford PEG-PSQ-2. The synthesis of 0.2 μM syringe filters to remove undigested cellular material. The
H2N-PEG(5K) has been reported in the literature.[
4g]
amount of Gd(III) incorporated with each cell pellet was then deter-
Synthesis of Anisamide-Modified PEG-PSQ-2 (AA-PEG-PSQ-2) : mined by ICP-MS.
The synthesis of anisamide-modified PEG-PSQ-2 was carried out
via a coupling reaction of PSQ-2 (10 mg) in dry acetonitrile, using
1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC, 50 mg)
as coupling agent, with 100 mg of a mixture of H N-PEG(5K) and
2
anisamide-functionalized PEG-NH (5K) (70:30 wt/wt) for 24 h Supporting Information
2
at room temperature. The synthesis of AA-PEG-NH (5K) has been
2
reported in the literature.[4g]
Supporting Information is available from the Wiley Online Library
Synthesis of FITC-Labeled PSQ-2 Nanoparticles: To prepare or from the author.
FITC-PSQ-2 particles, a FITC-silane derivative (FITC-AP-TES) was
first prepared. Briefly, a solution of FITC-AP-TES was prepared by
adding 6.8 mg (0.0175 mmol) of FITC in 1.1 mL of dry DMSO; to Acknowledgements
this solution, 4.95 μL (4.65 mg, 0.0211 mmol) of 3-aminopropyltri-
ethoxysilane was added, and the reaction was stirred at room tem- This work was supported by the NCI (U01-CA151455 and U54-
perature in the dark for 6 h. The final solution was diluted to 3 mL 151652). The authors thank the staff of the Biomedical Research
3
530 www.small-journal.com
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2013, 9, No. 20, 3523–3531

Biodegradable Polysilsesquioxane Nanoparticles for MRI
Imaging Center at UNC-Chapel Hill for help with MRI studies. JLV-E
thanks the Carolina Postdoctoral Program for Faculty Diversity for
a postdoctoral fellowship. WJR was a National Science Foundation
predoctoral fellow. HL acknowledges the HHMI-FSC for summer
research fellowships.
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Received: January 19, 2013
Revised: March 11, 2013
Published online: April 24, 2013
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small 2013, 9, No. 20, 3523–3531
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