Multi-chromatic pH-activatable 19F-MRI nanoprobes with binary ON/OFF pH transitions and chemical-shift barcodes

Article abstract of DOI:10.1002/anie.201301135

Imaging all the people: Using ionizable diblock copolymers a series of nanoprobes encoded with different ¹⁹F reporters for specific pH transitions is prepared for use in MRI. The pH response of the nanoprobes is extremely sharp (ΔpH_ON/OFF≈0.25 pH), and results from the disassembly of polymer micelles (see scheme). A collection of three nanoprobes provides the proof of concept and allows for a qualitative measurement of environmental pH values. Copyright

Full text of DOI:10.1002/anie.201301135

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Communications
DOI: 10.1002/anie.201301135
Imaging Agents
Multi-Chromatic pH-Activatable 19F-MRI Nanoprobes with Binary
ON/OFF pH Transitions and Chemical-Shift Barcodes**
Xiaonan Huang, Gang Huang, Shanrong Zhang, Koji Sagiyama, Osamu Togao, Xinpeng Ma,
Yiguang Wang, Yang Li, Todd C. Soesbe, Baran D. Sumer, Masaya Takahashi, A. Dean Sherry,
and Jinming Gao*
Magnetic resonance imaging (MRI) is a powerful non-
invasive imaging technique that has greatly impacted basic
biological research as well clinical diagnosis of cancer and
other diseases.[1] Conventional MR contrast agents are T1 (e.g.
Gd-DTPA) or T2-based (e.g. iron oxide), which cause
significant longitudinal or transverse relaxation of protons,
respectively.[2] Despite their success in many biological
applications, one potential limitation is the lack of multi-
chromatic features that allows for simultaneous detection of
multiple signals. Recently, 19F has received significant atten-
tion in MR imaging and spectroscopy studies.[3] Compared to
1H-MRI, 19F-MRI has little biological background owing to
the low levels of endogenous fluorine in the body. Moreover,
19F has 100% natural abundance and its gyromagnetic ratio
cell membrane is observed in cancer cells compared to normal
cells.[11] A variety of different types of MRI agents have been
reported for measuring pH values,[12] but all have a rather
broad pH response which may limit the accuracy of the pH
measurement, particularly when the pH perturbation in the
pathological tissue is small. Moreover, it is often necessary to
administer another pH-insensitive agent to correct for the
contribution of agent concentration to obtain pH-sensitive
signals, which makes the procedure complicated and difficult
to perform.[13]
Herein we describe the development of pH-sensitive 19F-
MRI nanoprobes with a binary (ON/OFF) response to
a specific, narrow pH transition (0.25 pH unit). We theorize
that a collection of such nanoprobes where each pH transition
is encoded with a specific 19F signature will allow for a simple
readout of environmental pH value through an “activation
barcode”. To demonstrate this proof of concept, we synthe-
sized three 19F-MRI nanoprobes with different pH transitions
and 19F-reporters (Scheme 1). Through these nanoprobes, we
show in phantom studies the feasibility of using either 19F
NMR spectroscopy or imaging to discriminate the pH differ-
1
(40.06 MHz/T) is second only to H, which makes it more
sensitive for detection over other nuclei.[3f]
Herein, we report on the development of “multi-colored”
pH-activatable 19F-MRI nanoprobes with tunable pH tran-
sitions. Recently, extensive efforts have been dedicated to the
development of stimuli-responsive nanoprobes.[4] Various
nanosystems that respond to pH,[5] enzymatic expression,[6]
redox reaction,[7] temperature,[8] and light[9] have been
reported. Among these stimuli, the pH stands out as an
important physiological parameter that plays a critical role in
both the intracellular (pHi) and extracellular (pHe) milieu.[10]
For example, dysregulated pH was described as another
hallmark of cancer, where a “reverse” pH gradient across the
[*] Dr. X. Huang, Dr. G. Huang, Dr. X. Ma, Dr. Y. Wang, Y. Li, Prof. J. Gao
Department of Pharmacology, Harold C. Simmons Comprehensive
Cancer Center, UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvds, Dallas, TX 75390 (USA)
E-mail: jinming.gao@utsouthwestern.edu
Dr. S. Zhang, Dr. K. Sagiyama, Dr. O. Togao, Dr. T. C. Soesbe,
Prof. M. Takahashi, Prof. A. D. Sherry
Advance Imaging Research Center
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvds, Dallas, TX 75390 (USA)
Dr. B. D. Sumer
Scheme 1. a) pH-activatable ON/OFF 19F-MRI nanoprobes from ioniz-
able diblock copolymers. At pH>pKa, the hydrophobic segments self-
assemble into a micelle core leading to 19F signal suppression as
a result of restricted polymer chain motion. Upon pH activation
(pH<pKa), micelle disassembly leads to dissociated unimers and
a strong 19F signal. b) Structural formula of three representative
diblock copolymers containing different pH responsive segments and
19F reporter moieties, their pKa values and 19F chemical shifts (in ppm,
relative to trifluoroacetic acid, or TFA), respectively, are shown in
parenthesis.
Department of Otolaryngology, UT Southwestern Medical Center at
Dallas, 5323 Harry Hines Blvds, Dallas, TX 75390 (USA)
[**] This work is supported by the NIH (R01CA129011, R01EB013149).
We acknowledge the assistance of the Southwestern Small Animal
Imaging Resource, which is supported in part by NCI U24
CA126608, the Simmons Cancer Center through an NCI Cancer
Center Support Grant (P30 CA142543).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301135.
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ences in the microenvironment (i.e. pH = 7.4, 6.5, 5.5, and
4.5).
higher molar fraction of TFE (Figure S2e). Data show the T2
is relatively unchanged (over 40 ms) when the TFE content is
below 20 mol%. Based on these data, we chose 20 mol% (i.e.
PEO-b-P(DPA48-r-TFE12) as the optimal 19F-reporter compo-
sition in subsequent pH response studies.
The challenge in designing a set of multi-colored pH-
activatable 19F-nanoprobes is two-fold: first is the availability
of reporter molecules that can be distinguished by MRS/I. For
1
this purpose, 19F is highly advantageous over H probes as
19F NMR spectra of PEO-b-P(DPA48-r-TFE12) copolymer
collected as a function of pH demonstrate ultra-pH respon-
sive behavior (Figure 1), similar to previously reported
many 19F reporter molecules have diverse chemical shifts and
narrow peak widths that can be easily differentiated. The
second is to devise an activation mechanism in which the
signal intensities of these 19F reporter molecules are highly
responsive to the pH changes in the environment. In this
regard, we adopted a strategy of using changes in spin–spin
relaxations between the micelle and unimer states to turn
ON/OFF 19F signals in response to the pH value.[3e,i] 19F
reporters are introduced to the ionizable block (PR) of
amphiphilic copolymers consisting of a hydrophilic poly(eth-
ylene oxide) (PEO) segment and tertiary amine/ammonium
segment (Scheme 1b). We hypothesize that at pH > pKa,
hydrophobic micelle assembly results in highly restricted
chain motions and short spin–spin relaxation times (T2!0) to
effectively broaden and eliminate the 19F signals; at pH < pKa,
protonation of ammonium groups will result in micelle
disassembly, conformational flexibility in the dissociated
polymer chains (unimers), and reappearance of the previous
19F signal.
Figure 1. a) 19F spectra of 2 mgmLꢀ1 PEO-b-P(DPA48-r-TFE12) micelles
in deuterated acetate buffers at different pH values. TFA was used as
an external reference with its chemical shift set as 0. b) Normalized 19
F
signal intensity as a function of pH value. Data was obtained from (a).
For initial development, we synthesized the poly-(ethyl-
ene oxide)-b-poly[2-(diisopropylamino) ethyl methacrylate-r-
trifluoroethyl methacrylate] (PEO-b-P(DPA-r-TFE)) copo-
lymer using atom transfer radical polymerization method.[14]
To investigate the optimal composition, we synthesized
a series of PEO-b-P(DPA-r-TFE) copolymers with increasing
molar ratios (5 to 75 mol%) of the TFE component
(Table S1–S2, Figure S1 in the Supporting information). A
higher TFE content should lead to more intense 19F signals,
whereas too much TFE may override the pH response from
the DPA segment and induce micelle aggregation even at low
pH values. Gel permeation chromatography (GPC) and
1H NMR characterization demonstrated that all the copoly-
mers had similar molecular weights (1.5–1.8 ꢀ 104 Da) and
polydispersity (Table S1, Figure S1). pH titration of the
copolymers showed that the TFE content had a considerable
influence on the pKa and pH response of the copolymers. At
5 mol% of TFE, the pKa is 6.3, similar to the PEO-b-PDPA
copolymer without TFE.[5c] An increase in TFE content
decreased the pKa of the copolymers (Figure S2a). Based on
these pKa values, we chose pH 4.0 (below the pKa values of all
the copolymers) to evaluate the effect of TFE content on the
19F signal intensity (dF = 2.3 ppm for TFE relative to TFA).
The 19F signal intensity as a function of TFE content showed
a bell-shaped response curve, where it reached a maximum at
40 mol% TFE. At pH 4.0, dynamic light scattering (DLS)
experiments showed that all the copolymers except the PEO-
b-P(DPA16-r-TFE44) (73 mol%) were in the unimer state as
indicated by their small size (under 10 nm in diameter;
Figure S2c). Whereas the PEO-b-P(DPA16-r-TFE44) copoly-
mer formed micelles with a hydrodynamic diameter of 44 nm
despite most of the amino groups being protonated at this
pH value. The decrease of 19F intensity can be explained by
the rapid increase of spin–spin relaxation (or decreased T2) at
fluorescent nanoparticles.[5c,d] Below pH 6.0, we observed
complete activation of 19F signals; above pH 6.2, the 19F
signals largely disappeared. The pH difference (DpH10–90%
)
between 10 to 90% signal difference is 0.25 pH units. This
ultra-pH response is a unique property of this class of
ionizable amphiphilic block copolymers, where hydrophobic-
ity-driven micellization dramatically increased the coopera-
tive deprotonation of the ammonium blocks.[5c,d] Transmission
electron microscopy (TEM) of PEO-b-P(DPA48-r-TFE12)
verified the formation of micelles at pH 7.4 (above its pKa
of 6.1) and complete micelle dissociation at pH 5.0 (Fig-
ure S3a). The micelle–unimer transition was further corrobo-
rated by 1H NMR spectroscopy (Figure S3b) and dynamic
light scattering, which showed the hydrodynamic diameters
were changed from 40 to 6 nm at pH 7.4 and 5.0, respectively
(Figure S3c).
To investigate the ON/OFF pH-activatable MR imaging
capability of the nanoprobes, we prepared a sample in two
concentric tubes where both tubes were filled with PEO-
P(DPA48-r-TFE12) at 25 mgmLꢀ1 but the pH values of the
inner and outer tubes were set at 5.0 and 7.4, respectively.
Axial 1H MRI images showed two compartments with similar
signal intensities (left panel, Figure 2a). In contrast, the
corresponding 19F MRI images showed an intense signal (ON)
in the inner tube but no signal (OFF) in the outer tube (right
panel, Figure 2a). We quantified the signal intensity in
different regions of interest (ROI) over the background
noise (Figure 2b). At 55 min, the 19F signal-to-noise ratio
(SNR) reached 31-fold for the PEO-b-P(DPA48-r-TFE12)
nanoprobes at pH 5.0 (ON state). Then we compared the
contrast of 19F images between the ON and OFF states at
pH 5.0 and 7.4, respectively. The contrast ratio (SNRpH5.0
/
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pattern of nanoprobe activation. More specifically, the (000)
solution corresponds to the solution at pH 7.4, in which all the
nanoprobes were OFF. Accordingly, the (001), (011), and
(111) solutions correspond to solutions with pH values at 6.5,
5.5 and 4.5, respectively. The nanoprobe barcodes successfully
distinguished the solution pH values. Lastly, addition of fetal
bovine serum (5 or 10%) to the nanoprobe solutions at
pH 4.5 did not affect the signal contrast significantly, demon-
strating successful 19F detection in biologically relevant media
(Figure S5).
In addition to 19F spectroscopy, we also used 19F MRI to
spatially resolve the nanoprobe activation map. A phantom
sample was prepared in which four smaller tubes (each
containing the same nanoprobe mixture in solutions at
pH 7.4, 6.5, 5.5, and 4.5) were placed in a bigger tube with
1
water only. T1-weighted H MRI images show similar signal
Figure 2. a) 1H and 19F MRI images of PEO-b-P(DPA48-r-TFE12)
(25 mgmLꢀ1) phantom at pH 5.0 (inner tube) and 7.4 (outer tube).
b) SNR of 19F signals for PEO-b-P(DPA48-r-TFE12) as a function of
scanning time at pH 5.0 (left panel) and comparison of SNR ratios at
intensity from all the tubes and the surrounding water
(Figure 3c). For 19F MR imaging, we selectively activated
each 19F reporter at its chemical shift to examine the
nanoprobe activation. Based on results from each 19F channel,
we were able to obtain the barcode information for the
different regions of interest (Figure 3c). Potentially, by
combining the 19F spectroscopy and imaging capabilities, we
can generate a pH map where each voxel can be encoded with
an activation barcode to indicate its environmental pH value
with spatial discrimination.
1
pH 5.0 and 7.4 from both H and 19F MRI images (right).
SNRpH7.4) is 27-fold based on the 19F images, demonstrating
that the 19F reporters on the polymers are highly responsive to
the pH changes in the environment. In comparison, the
SNRpH5.0/SNRpH7.4 ratio from the 1H images was only 1.2.
Finally, we investigated the “barcode” concept using
a mixture of 19F-MRI nanoprobes with different pH transi-
tions and 19F reporter molecules to distinguish pH values in
the microenvironment. In addition to TFE (dF = 2.3 ppm), we
introduced two additional 19F reporter molecules (Scheme 1b,
DFB and BTFB, dF = ꢀ33.2 and 13.0 ppm, respectively).
These reporter molecules were incorporated into two new
copolymers with different pH sensitivities, poly(ethylene
oxide)-b-poly[2-(pentamethylene imino) methacrylate-r-2-
(methacryloyloxy) ethyl 3,5-bis(trifluoromethyl) benzoate]
(PEO-b-P(C6A-r-BTFB)) and poly(ethylene oxide)-b-
poly[2-(dibutylamino) methacrylate-r-2-(methacryloyloxy)
In summary, we report the feasibility of a series of
multichromatic pH-activatable 19F nanoprobes encoded with
different 19F reporters at specific pH transitions. Compared to
small molecular pH sensors (typically 2 pH unit for a 10-fold
signal change across pKa), the pH response of these nanop-
robes is extremely sharp (DpHON/OFF ꢁ 0.25 pH unit) and can
be used as binary indicators for a specific pH transition. The
current three nanoprobe collection provides the proof of
concept and allows for a qualitative measurement of environ-
mental pH values. This nanoplatform can potentially over-
come the instrument complexity and short T1 limitation of the
13C-based hyperpolarization probes.[15] Moreover, compared
to chemical exchange saturation transfer (CEST) or 1H agents
with which small pH-dependent chemical shifts are quanti-
fied,[12c,16] the chemical shifts of 19F reporters are widely
separated and easily differentiated for binary readout and
data processing. Development of additional nanoprobes with
more refined pH transitions will be useful to narrow the pH
transitions and improve the precision of the pH measurement.
In addition, use of hybrid nanoparticles to include all 19F-
encoded polymers in one system could further unify pharma-
cokinetics and biodistribution during in vivo studies. Through
a barcode map from 19F-imaging spectroscopy, it is conceiv-
able to generate a pH map in three dimensions. Along with
these exciting potentials, one main challenge in subsequent
preclinical translation of these nanoprobes is the relatively
low detection sensitivity of 19F-MRS/I. Optimization of MR
scan time, pulse sequence or coil design should further
improve the current detection limit (0.16 mgmLꢀ1 19F). Image
resolution can also be compromised to achieve higher
detection sensitivity. Upon successful demonstration, the 19F
nanoprobes will add to the existing arsenal of pH sensors to
measure tissue pH values, an important physiological param-
ethyl
3,5-difluorobenzoate]
(PEO-b-P(DBA-r-DFB);
Table S3). pH titration experiments demonstrated similar
ultra-pH responsive properties of the two new copolymers
(Figure S4). The pKa values of the PEO-b-P(C6A-r-BTFB)
and PEO-b-P(DBA-r-DFB) copolymers were 7.0 and 5.0,
respectively, in addition to PEO-b-P(DPA-r-TFE) (pKa =
6.1). Based on these pKa values, we defined a three-digit
barcode where each digit corresponds to one nanoprobe (with
pKa from low to high), and has a binary response (1 for ON, 0
for OFF). For better visual demonstration, we also assigned
a single color to each nanoprobe for the ON state (black for
the OFF state). Such a barcode design allows for the direct
readout of the microenvironment pH value within two
adjacent pKa values in which one nanoprobe is ON and the
other is OFF (Figure 3a).
To validate this concept, we performed a double blind
experiment, in which four solutions at pH 7.4, 6.5, 5.5, and 4.5
were first prepared containing the same mixture of the three
nanoprobes. 19F spectroscopy was then performed for each
solution. Figure 3b shows a clearly distinguished barcode
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Figure 3. a) Schematic illustration of the activation barcode concept for direct
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F
spectra of a mixture of three PEO-b-P(R-r-F) nanoprobes in acetate buffers of
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Keywords: 19F MRI · imaging agents · micelles · pH imaging ·
polymers
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