Reduction Triggered in Situ Polymerization in Living Mice

Article abstract of DOI:10.1021/jacs.0c07594

"Smart"biomaterials that are responsive to physiological or biochemical stimuli have found many biomedical applications for tissue engineering, therapeutics, and molecular imaging. In this work, we describe in situ polymerization of activatable biorthogonal small molecules in response to a reducing environment change in vivo. We designed a carbohydrate linker- and cyanobenzothiazole-cysteine condensation reaction-based small molecule scaffold that can undergo rapid condensation reaction upon physiochemical changes (such as a reducing environment) to form polymers (pseudopolysaccharide). The fluorescent and photoacoustic properties of a fluorophore-tagged condensation scaffold before and after the transformation have been examined with a dual-modality optical imaging method. These results confirmed the in situ polymerization of this probe after both local and systemic administration in living mice.

Full text of DOI:10.1021/jacs.0c07594

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Article
Reduction Triggered In Situ Polymerization in Living Mice
Lina Cui, Sandro Vivona, Bryan R. Smith, Sri Kothapalli, Jun Liu, Xiaowei Ma, Zixin Chen,
Madelynn Taylor, Paul H Kierstead, Jean M. J. Frechet, Sanjiv S. Gambhir, and Jianghong Rao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c07594 • Publication Date (Web): 17 Aug 2020
Downloaded from pubs.acs.org on August 18, 2020
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Reduction Triggered In Situ Polymerization in Living Mice
Lina Cui1,2,3,4, Sandro Vivona5,6,7, Bryan Ronain Smith3,†, Sri R. Kothapalli3,#, Jun Liu1,2, Xiaowei Ma1,2,
Zixin Chen1,2,4, Madelynn Taylor3, Paul H. Kierstead8, Jean M.J. Fréchet8, Sanjiv S. Gambhir3,9,10,‡ and
Jianghong Rao3,4,*
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1Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA; 2UF Health Cancer Center, University of
Florida, Gainesville, FL, USA; 3Molecular Imaging Program at Stanford, Bio-X Program, Department of Radiology, School
of medicine, Stanford University, Stanford, CA, USA; 4Department of Chemistry, Stanford University, CA, USA; 5Department
of Molecular and Cellular Physiology, Stanford University, CA, USA; 6Department of Structural Biology, Stanford University,
Stanford, CA, USA; Department of Photon Science, Stanford University, Stanford, CA, USA; College of Chemistry,
University of California, Berkeley, CA, USA; 9Department of Bioengineering, Stanford University, CA, USA; 10Department
of Materials Science and Engineering, Stanford University, Stanford, CA, USA.
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ABSTRACT: “Smart” biomaterials that are responsive to physiological or biochemical stimuli have found many biomedical
applications for tissue engineering, therapeutics and molecular imaging. In this work we describe in situ polymerization of activatable
biorthogonal small molecules in response to reducing environment change in vivo. We designed a carbohydrate linker- and
cyanobenzothiazole-cysteine condensation reaction-based small molecule scaffold that can undergo rapid condensation reaction upon
physiochemical changes (such as a reducing environment) to form polymers (pseudopolysaccharide). The fluorescent and
photoacoustic properties of fluorophore-tagged condensation scaffold before and after the transformation have been examined with
a dual-modality optical imaging method. These results confirmed the in situ polymerization of this probe after both local and systemic
administration in living mice.
Introduction
Biomaterials that can undergo in situ transformation with
exposure to specific biomarkers or environmental cues in living
subjects can potentially lead to new biomedical applications.1
For example, preformed polymers that can cross-link or solidify
in vivo after injected locally when exposed to light,2-3
temperature,4 pH changes,5 enzymes,6-7 or presence of specific
ions8 can provide better tissue-material contact and can be used
for facilitating local transplantation of medical scaffolds,9 tissue
engineering,10-11 drug delivery12-13 and imaging.14 Compared to
polymeric materials, small molecules possess properties of
efficient tissue distribution, deep penetration, and fast systemic
clearance in normal tissues. However, small molecules also
suffer from poor retention compare to polymeric material. Thus
in situ self-assembly of small molecules into large
macromolecules or polymeric materials at target sites can take
advantage of the efficient distribution and clearance of small
molecules as well as the enhanced retention of macromolecules
and offer unique opportunities in designing new materials for
diagnostics and therapeutics.15-17
One of the strategies for assembling small molecules into
macromolecules inside complex biological environment is
through covalent bonding enabled by bioorthogonal reactions.18
These reactions such as ketone/hydroxylamine condensation,19-
22
copper free azide-alkyne cycloaddition,23-26 Staudinger
Figure 1. Structures of molecular probes used in the study and their
activation to polymers. a. Condensation reaction between
ligation27-29 and tetrazine-trans-cyclooctene cycloaddition,30-36
have been applied to site-directed protein labeling,37 studying
cyanobenzothiazole and cysteine (a.k.a. CBT Condensation). b. Probes 1
38-39
metabolic pathways24,
and engineering polymeric
and 3 undergo polymerization upon reductive activation. Control probes 2
and 4 remain as monomers under the same conditions.
biomaterials.40-42 Their applications in “smart” biomaterials that
are responsive to physiological or biochemical stimuli are
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limited due to the fact that most of the chemical moieties used cyclization and provide an attractive strategy for in situ
1
2
3
4
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8
in these reactions cannot be biochemically masked. Recently
there have been some reports on using light or enzymes to
modulate bioorthogonal reactions,43-52 but not yet demonstrated
in vivo. For the past few years, we have been studying the
reaction between aminothiols and aromatic nitriles53-56 and its
applications for in vivo molecular imaging.57-63 One of the most
attractive features of this reaction is that its reactivity can be
modulated by pH, reducing environment or enzyme activities
by masking the aminothiol with different responsive moieties.
One of the fastest reactions we’ve tested so far is between the
cyanobenzothiazole (CBT) group and free cysteine, which has
a second-order rate constant of 9.19 M-1s-1.53 We and others
have used this reaction for protein labeling,64-67 formation of
hydrogels,53, 68-69 fluorescence lifetime imaging70 and in vivo
imaging of protease activity.53, 57-58 This condensation reaction
therefore holds great potential for constructing “smart”
molecules that can go through in situ transformation into large
macromolecules or polymeric materials when encounters
specific biological stimuli.
formation of hydrogels from small molecules (Fig. 1). The
carbohydrate moieties are used extensively in nature to form
rigid polymers, such as cellulose. The equatorial conformation
of glucose in cellulose provides rigidity for the linear
polysaccharides. Here we report a carbohydrate-based small
molecule scaffold that can undergo CBT-cysteine condensation
to form polymers under reducing conditions. We used a dual-
modality optical imaging method to measure the fluorescence
and photoacoustic properties (conversion of non-ionizing
radiation to pressure/ultrasound waves)71-73 of new scaffold
based fluorescent probes before and after the polymerization
and demonstrated the in situ formation of polymers in living
mice.
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Results and discussion
Design and synthesis of polymerizable molecular scaffold.
To evaluate the polymerization reaction, we synthesized two
sets of probes with D-glucosamine as the backbone to facilitate
the polymerization (Fig. 1). The CBT is linked to C-6 on the
glucosamine via click reaction and cysteine is linked to the C-2
amine via a peptide linker. The fluorescent probes 1 and 2
contained an IR800 dye as an imaging moiety to detect their
fluorescence and photoacoustic properties. IR800 dye was
selected for its suitable excitation/emission wavelength (774
nm/789 nm) for in vivo imaging, its large extinction coefficient
Our previously reported CBT conjugates tend to form cyclic
dimers or oligomers after condensation instead of forming
linear polymers due to the flexible peptide linkers within the
scaffolds.53, 57 We hypothesized that a rigid linker in the scaffold
would promote the elongation of the linear polymers over
Figure 2. Characterization of polymerization. a. Comparison of elution profiles of the polymerization reaction of probe 3 at various concentrations (1, 10,
100 μM, as indicated) after different reactions times (10, 30, 60, 120, 240, 480 min, as indicated). The molecular weights of peaks at TE = 14.8 min and 17.7
min are 25 kDa and 1 kDa respectively (Supporting Fig. S4); the peak at 16.5 min contains trimers and higher-order oligomers as detected by MALDI-MS
(Supporting Fig. S6). b. 2D intensity contour image of the elution profiles at indicated concentrations and reaction time points. c, d, e. Products population
of the reaction at 1, 10, and 100 μM vs. time, respectively.
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(240,000 M-1 cm-1), and its good water solubility. The other set
of probes and contained 2,2’,2’’-(10-(2-((2,5-
dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid
and hydrodynamic radius of 3 nm (Supporting Fig. S2) in 10
1
2
3
4
5
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7
8
3
4
a
minutes, together with monomer 3, dimer (Supporting Fig. S2
peak 4, S5), oligomers (Supporting Fig. S2 peak 3, S6) and a
second portion of polymers of around 20 kDa (Supporting Fig.
S2 peak 2, S4) as detected by matrix-assisted laser
desorption/ionization mass spectroscopy (MALDI-MS). To
evaluate the polymerization at low concentrations, probe 3 was
activated at 1, 10 and 100 μM by GSH (10 mM), and the
polymerization profiles were monitored from the initial 10
minutes to 8 hours after incubation in reaction buffer (Fig. 2).
Formation of polymers was concentration-dependent, with
larger polymer species formed at higher concentrations (Fig. 2a,
2b). At 100 μM, nearly 80% of probe 3 was converted to
polymers at around 25 kDa (~25 repeating units) within the first
1 hour of reaction (Fig. 2a, 2b, 2e). At concentrations of 10 μM
or even 1 μM, around 40% of probe 3 formed polymer species
at around 25 kDa within 1 hour (Fig. 2a, 2b, 2c, 2d). These in
vitro results demonstrated the efficiency of the polymerization
reactions at various concentrations. To our delight, this
polymerization can proceed efficiently at as low as a few micro
molar concentrations, making it attractive for applications in
living systems.
(DOTA)
moiety for its water solubility and its potential to chelate Gd3+
for magnetic resonance imaging (MRI) or radioactive metal
ions such as Cu-64 or Ga-68 for positron emission tomography
(PET) imaging in the future. Probes 1 and 3, containing a
disulfide linkage on the cysteine, can be activated by reducing
environment to generate the free N-terminal cysteine group that
then reacts with CBT group on the other molecule to initiate the
polymerization (Fig. 1). The control probes 2 and 4 cannot
expose the thiol group in reducing environment and should
remain as small molecules.
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A convergent synthetic scheme was developed to obtain all
probes 1-4 (Supporting Information Fig. S1). The synthesis
started from free D-glucosamine, and an azido group was linked
to the C-1 position to lock its chair conformation. A cysteine
group was then conjugated to the amino group, and the CBT
group was introduced at the C-6 position via a copper-catalyzed
azide-alkyne cycloaddition (CuAAC). Both IR800 and DOTA
were introduced via their NHS esters.
Optical properties of small molecule probes upon in situ
polymerization. We evaluated the optical properties of probes
1 and 2 before and after activation by glutathione (Fig. 3,
Supporting Fig. S7). Glutathione plays a key role in regulating
the redox potential in mammalian cells and its concentration can
range from several μM in plasma to several mM in cells and
tumor tissues.74-80 Our group and others have demonstrated that
the CBT condensation reaction reactions can proceed under
physiological conditions where glutathione is present in cells as
well as in living animals.53, 58, 70, 81-83 Incubation of probe 1 (100
μM) in PBS buffer containing reducing agent glutathione (GSH,
10 mM) or tris(2-carboxyethyl)phosphine (TCEP, 10 mM) at
pH 7.4 resulted in a fluorescence signal drop by around 70%
Characterization of polymerization reactions using SEC-
MALS. The polymerization reactions were monitored by the
size exclusion chromatography (SEC) coupled with multiangle
light scattering (MALS) to analyze the sizes, mass and relative
distribution of the resulting polymers (Fig. 2, Supporting Fig.
S2). The activated or un-activated probe 3 was injected onto a
WTC-010S5 column, and the elution profile was monitored by
light scattering at 658 nm and differential refractometry. The
triggered polymerization was very efficient in the presence of
GSH (10 mM): the majority (> 60%) of probe 3 (at a
concentration of 10 mM) was converted to polymers at a size of
around 40 kDa (~40 repeating units, Supporting Fig. S2 peak 1)
Figure 3. Characterization of polymerization using fluorescence and photoacoustic signals. a. White light (upper) and fluorescence (lower) images of
(1) probe 2 (100 μM) in PBS buffer at pH 7.4, (2) probe 2 (100 μM) in PBS buffer containing TCEP (10 mM) at pH 7.4, (3) probe 2 (100 μM) in PBS buffer
containing GSH (10 mM) at pH 7.4, (4) probe 1 (100 μM) in PBS buffer at pH 7.4, (5) probe 1 (100 μM) in PBS buffer containing TCEP (10 mM) at pH
7.4, (6) probe 1 (100 μM) in PBS buffer containing GSH (10 mM) at pH 7.4. b. Silver nitrate staining (left) and fluorescence image (right) of gel
electrophoresis (25% acrylamide gel) of (1) probe 2 (6 nmol), (2) probe 2 (0.6 nmol), (3) probe 2 (0.06 nmol), (4) probe 1 (6 nmol), and (5) probe 1 (0.6
nmol) in loading buffer containing GSH (10 mM) at pH 7.4. c. Quantification of fluorescence signals of probe 1 (100 μM) in PBS buffer at pH 7.4, probe 1
(100 μM) with GSH (10 mM) at pH 7.4, and probe 2 (100 μM) with GSH (10 mM) at pH 7.4 after 30 min incubation. d. Fluorescence changes of probe 1
at 0.1, 1, 10 μM and probe 2 at 10 μM over two hours at pH 7.4. e. Fluorescence images of sealed PE tubes of (1) and (2) probe 1 (100 μM) in PBS buffer
at pH 7.4, (3) and (4) probe 1 (100 μM) in PBS buffer containing GSH (10 mM) at pH 7.4, (5) and (6) probe 2 (100 μM) in PBS buffer containing GSH (10
mM) at pH 7.4. (7) and (8) probe 1 (100 μM) in PBS buffer at pH 7.4, (9) and (10) probe 1 (100 μM) in PBS buffer containing GSH (10 mM) at pH 7.4,
(11) and (12) probe 2 (100 μM) in PBS buffer containing GSH (10 mM) at pH 7.4. f. Processed photoacoustic images (3D maximization) of the same
sample tubes (1)-(6) in e. g. Quantification of photoacoustic signals of probe 1 (100 μM) in PBS buffer at pH 7.4, probe 1 (100 μM) with GSH (10 mM) at
pH 7.4, and probe 2 (100 μM) with GSH (10 mM) at pH 7.4. Standard deviations are shown by error bars.
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within 30 min (Fig. 3a, 3c) likely due to the close proximity of fluorescence signal upon the formation of polymers.
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IR800 dye and self-quenching after polymerization; in
comparison, probe 2 under the same conditions or probe 1
without incubation with GSH or TCEP barely showed any
changes in fluorescence signals within two hours (Fig. 3a, 3c).
Electrophoretic gels of the GSH-treated probe 1 and 2 were
stained using silver nitrate (Fig. 3b) and scanned using Maestro
hyperspectral fluorescence imaging system (PerkinElmer,
635±25 nm excitation filter, 675 nm long-pass emission filter,
image acquisition from 670 to
Imaging polymerization in living animals. We next
evaluated the polymerizable probe with both fluorescence and
PA imaging in a model system in living mice. The back of
BALB/c mice was shaved, and a solution of GSH was injected
subcutaneously on the furless back to form two spots of
reducing environment (Fig. 4a). Probe 1 (1 nmol in 30 μL of
PBS buffer) and probe 2 (1 nmol in 30 μL of PBS buffer) were
9
900 nm). From the silver
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staining, control probe 2 showed
single bands at all concentrations
(6, 0.6, and 0.06 nmol per 6 μL
loaded) tested; while probe 1 (6
nmol per 6 μL loaded) showed
smeared bands at a position
similar to that of the 25-40 kDa
fraction from SEC (Fig. 3b, left),
and
probe
1
at
lower
concentrations (0.6 nmol per 6
μL loaded) produced slightly
smaller polymer species (Fig. 3b,
left). Interestingly, in comparison
to the fluorescence intensity of
probe 2 at the test concentrations,
probe 1 only gave a major
fluorescence band of the dimer
(detected by MALDI-MS after
gel extraction), however the
larger polymer species remained
dark due to self-quenching of
IR800 dye after polymerization
occurred (Fig. 3b, right).
Photoacoustic (PA) imaging
detects the acoustic signals
generated during thermoelastic
expansion
due
to
the
photoabsorption. Therefore, PA
imaging offers deeper tissue
penetration and higher spatial
resolution than fluorescence
imaging.71-73 PA signals were
measured using a Vevo LAZR
instrument (Visualsonics, 2100
High-Resolution
Imaging
System). Probe 1 and 2 (each at
100 μM) with or without GSH
(10 mM) were sealed in
polyethylene tubes and buried in
agarose gel, and fluorescence
imaging were performed 30 min
after the reaction started,
immediately before acquiring
photoacoustic signals (Fig. 3e, Figure 4. Fluorescence and photoacoustic imaging of polymerizable probe in living mice. a. Fluorescence
3f). While GSH-incubated probe
1 showed reduced fluorescence
signal compared with probe 2
(Fig. 4e), its PA signal increased
to nearly 200% of the signal from
probe 2 or probe 1 alone (Fig. 3f,
3g), suggesting the enhanced PA
effects accompanying reduced
imaging of subcutaneously injected probe 1 (right spot, 1 nmol) and 2 (left spot, 1 nmol) in living mice in 24
hours after injection of the probe. Area of injection was pre-treated with GSH. The images are presented at
different intensity scales to show the contrast of the two reaction spots. b. Ratio of fluorescence intensities of
probe 1 and 2 vs. time. Red symbols and line, fluorescence ratio of probe 1 and 2. c. First two hours of graph b.
d. Co-registered ultrasound and photoacoustic images of subcutaneously injected probe 1 (top, 1 nmol) and 2
(bottom, 1 nmol) in living mice in 24 hours after injection of the probe. Area of injection was pre-treated with
GSH. White scale bar in the ultrasound windows is 3 mm. The yellow dash-lined rectangular indicates the area
of interest for photoacoustic signals. e. Changes of photoacoustic intensities of probe 1 and 2 vs. time. Blue
symbols and line, probe 1; red symbols and line, probe 2. f. The first two hours of graph b. Standard deviations
are shown by error bars. **, P<0.0001.
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then injected to each of the two
1
2
3
4
reducing sockets. As expected, the
two probes initially displayed the
same fluorescence intensity, but
the fluorescence of probe
decreased drastically
1
in
5
comparison to probe 2 within the
first 30 minutes after injection
(Supporting Fig. S8). Interestingly
while the absolute fluorescence
signal decreased in both spots due
to the probe diffusion from the
inoculation site, probe 1 appeared
brighter than probe 2 afterwards
even with self-quenching: the
intensity ratio of probe 1 and 2
started to escalate, and reached
around 15 after 24 hours (Fig. 4b,
4c), indicating the prolonged
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retention of probe
activation.
1
upon
PA imaging (co-registered with
ultrasound imaging) was similarly
performed using the same model
system, and each spot was
monitored for 24 hours after probe
injection (Fig. 4d). Different from
fluorescence imaging, which
showed
drastic
fluorescence
decrease of probe 1 in the initial 30
min, PA signal of the same spot
increased 30 min after probe 1 was
injected (Supporting Fig. S9).
Comparing to probe 2, which
showed gradual decrease in PA
signal over time, probe 1 showed
initial PA enhancement in the first
hour after injection, followed by a
slow decline phase (Fig. 4e, 4f).
Together with the fluorescence
imaging results, the initial decrease
in fluorescence and increase in PA
signal resembled the trend in the in
vitro experiments (Fig. 3), and
probe 1 was retained longer in
tissue than probe 2, suggesting the
high probability of polymer
formation.
Imaging of polymerization in
tumor
models.
The
ideal
polymerizable small molecules
when injected systemically should
Figure 5. Fluorescence and photoacoustic imaging of polymerizable probe in tumor models. a.
Longitudinal fluorescence imaging of tumor-bearing mice with i.v. injected probe 1 (20 nmol) in 8 hours.
be
activated
to
form Tumor (indicated by red arrow) was pre-treated with GSH to activate probe 1. b. Comparison of the average
tumor fluorescence radiance intensity of mice groups injected with probe 1 (20 nmol, GSH treatment, red circle,
macromolecular structures at the
target site (such as a tumor) in the
living subjects, while diffusing
away in other non-targeted tissues
to provide better imaging contrast.
To assess whether our molecular
scaffold can be activated at desired
sites after intravenous injection, we
evaluated the probes 1 and 2 in
mice xenografted tumor models.
n = 5; 20 nmol, saline treatment, blue square, n = 5) or probe 2 (20 nmol, GSH treatment, black diamond, n =
5) vs. time (24 hours). Standard deviations are shown by error bars. c. First four hours of graph b. d. PA and
US co-registered tumor images of mice with i.v. injected probe 1 and probe 2 (each at 20 nmol) before and one
hour after injection. Tumor was pre-treated with intratumoral injection of GSH. Anatomical position of the
tumor is indicated by red (US images) or white (PA images) arrows. A &B, US (A) and PA (B) images of target
area before injection. C & D, US (C) and PA (D) images of target area one hour after i.v. injection of probe. e.
Comparison of the average tumor PA intensity of mice groups injected with probe 1-activated (20 nmol, GSH
treatment, red circle, n = 5), probe 1-unactivated (20 nmol, saline treatment, blue square, n = 5), and probe 2
(20 nmol, GSH treatment, black diamond, n = 5) vs. time. f. First four hours of graph e. g. Tumor to muscle
ratio of the PA signal of the same mice groups in e. h. First four hours of graph g. All error bars indicate standard
deviation. **, P<0.0001; #, not significant.
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The ability to trigger the polymerization at desired sites was Probe synthesis. Chemical syntheses and characterizations
1
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evaluated in female nude mice bearing subcutaneous U87MG
tumors that were pre-treated with intratumoral injection of GSH
or saline. Probe 1 or 2 (20 nmol in 200 μL saline) was
administered to tumor-bearing mice via tail vein injection, and
longitudinal fluorescence and PA imaging of these mice were
performed (Fig. 5, Supporting Fig. S10). Since both probe 1 and
2 are not cell permeable, the polymerization will occur outside
tumor cells, depending on the intratumoral delivery of GSH. We
hypothesized that formed polymers would diffuse away slower
than the free probe itself. The whole animal fluorescence
imaging showed the fluorescence of probe 1 accumulated in
GSH-pretreated tumor during the first one hour after probe
injection and declined slowly over the monitored time course of
24 hours (Fig. 5, Supporting Fig. S10). Probe 1 administered to
of probes 1-4 and intermediates are provided in Supporting
Information.
SEC-MALS
measurements.
Size
exclusion
chromatography (SEC) coupled with multiangle light scattering
(MALS) was performed by injecting 150 μg of activated or
unactivated probe 3 or 4 onto a WTC-010S5 column (Wyatt
Technology) previously equilibrated in PBS buffer at a flow
rate of 0.5 mL/min. The elution profile was monitored by light
scattering at 658 nm (HELEOS system, Wyatt Technology) and
differential refractometry (Optilab system, Wyatt Technology).
UV Absorbance was not used due to saturation of the detector
at the concentrations above the detection limit of light
scattering. Data analyses were carried out using ASTRA 6.0
software (Wyatt Technology). The refractive index increment
(dn/dc) value of 0.163 mLµg-1 was assumed based on
literature,84 as experimental determination was hindered by
limited solubility of samples. Calculated molecular weights
were further confirmed by MALDI-TOF mass spectrometry.
SEC traces of polymerization reactions of different
concentrations at different time points were plotted using Origin
Lab 8.5 as 3D stacking and 2D intensity graphs. Different
species (monomer, polymer species around 25 kDa alone or
with other oligomers) were quantified using integration of area
under curve (AUC) of the differential refractometry data and
plotted against time using Origin Lab 8.5.
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the mice bearing saline-pretreated tumor or probe
2
administered to the mice bearing GSH-pretreated tumor
produced much faster fluorescence signal decay after injection,
suggesting that probe 1 are 2 are poorly retained in tumor
specifically (Fig. 5b, 5c, Supporting Fig. S10).
PA imaging of each mouse at each specific time point was
performed immediately after the fluorescence scanning (Fig.
5d). PA signal in saline-pretreated tumors with probe 1
administration or GSH-pretreated tumors with probe 2
administration appeared close to the background level during
the monitored time course (Fig. 5d, middle and right, Fig. 5e,
5f); in comparison, the GSH-pretreated tumors with probe 1
administration gave strikingly high PA signals in the entire
time, peaking at 4 hours after probe injection (Fig. 5d, left, Fig.
5e, 5f). Importantly, the PA signal was nearly evenly distributed
throughout the tumor region. While examining the results of the
activated probe 1 in both of the PA imaging (Fig. 5d) and
fluorescence imaging (Fig. 5a), tumor PA signal peaked (at 4
hours post probe injection) later than its fluorescence signal (at
30 min to 1 hour post probe injection). Also, PA signal in tumor
remained high (nearly 50% of its peak value) even after 24
hours (Fig. 5e), but its corresponding fluorescence signal
dropped to around the same level as two other control
conditions after 4 hours (Fig. 5b, 5c). Together with the in vitro
results, remaining PA signal in tissue without fluorescence can
be attributed to the formation of polymers, which lose the
fluorescence via self-quenching.
Measurement of photoacoustic effects of in vitro samples.
PA imaging was performed using a LAZRTightTM imaging
enclosure coupled Vevo LAZR PA imaging system
(VisualSonics), equipped with a MS-250 linear array transducer
(21 MHz, 70% 6 dB two-way bandwidth, 256 elements) to
detect ultrasound signals, and a tunable Nd:YAG laser system
(OPOTEK, 680-950 nm, 20 Hz repetition rate, 5 ns pulse width,
50 mJ pulse peak energy) to generate optical pulses. For in vitro
measurements, the probe solutions were injected into one-inch
long polyethylene (PE100) tubing (Becton Dickinson, i.d. =
0.86 mm, o.d. = 1.52 mm), and the open ends of the tubes were
heat-sealed. The tubes were then inserted horizontally into a
freshly made semi-hardened agarose gel (1%) and the surface
of the gel was coated with even layer of agarose solution (1%,
~3 mm thick). PA imaging was then performed after the gel
completely solidified. PA signals were acquired using a 21 Hz
transducer at 40 dB PA gain in 3D mode at a wavelength of 774
nm (absorbance max of IR800 dye) and with an acquisition rate
of 5 frames/second. The 3D image of each sample tube was
processed using loaded Visualsonics Vevo software package,
and analyzed using Image J software (Fig. 3f, 3g).
Conclusions
Our previous experience with the CBT condensation
reactions often led to early cyclization and self-stacking to form
nanoparticles. Molecular scaffolds that can prevent the
formation of self-aggregates can further extend the application
of the scaffold and chemistry to other applications such as
therapeutics delivery and tissue engineering. In this work, we
explored a carbohydrate scaffold for its water solubility and
structural rigidity from the chair conformation to promote linear
polymerization. Our results have shown that linear
Fluorescence imaging of polymerizable probes in living
mice. Female BALB/c mice (4 months old, Charles River Inc.)
were anesthetized by isoflurane, and fur was removed to expose
the back skin (Fig. 4a). In the following day, the mice were
anesthetized and positioned on the heating platform inside a
polymerization can occur even at
a
few micromolar
Maestro
hyperspectral
fluorescent
imaging
system
concentration with this new rigid carbohydrate scaffold. To
address the challenge of the detection of polymerization in
living animals, we developed a dual-modality optical method
using fluorescence and PA imaging. With this imaging method,
we were able to demonstrate the in situ polymer formation with
reductive activation in living mice. Future efforts may extend
this system to other stimuli such as enzyme activation in tumors.
(PerkinElmer). GSH (20 mM in 20 μL of PBS buffer) was
injected subcutaneously under the back skin for each testing
spot, where probe 1 or 2 (each of 1 nmol in 30 μL of PBS buffer)
were then injected 5 min later. Fluorescence imaging was
performed immediately using the Maestro scanner (635±25 nm
excitation filter, 675 nm long-pass emission filter, images
acquisition from 670 to 900 nm). IR800 fluorescence was
deconvolved from tissue autofluorescence (Nuance v.3.0.1.2,
Experimental Section
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†B.R.S. is currently at the Department of Biomedical Engineering
PerkinElmer), and fluorescence intensity was quantified by
1
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4
5
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7
8
and Institute for Quantitative Health Science and Engineering,
Michigan State University.
region of interest (ROI) analysis (Fig. 4b, 4c).
PA imaging of polymerizable probes in living mice.
Female BALB/c mice were anesthetized and positioned on the
heating platform inside the LAZRTightTM imaging enclosure of
the Vevo LAZR imaging system. GSH (20 mM in 20 μL of PBS
buffer) was injected subcutaneously under the furless back skin
for each testing spot, where probe 1 or 2 (each of 1 nmol in 30
μL of matrigel) were then injected 5 min later. A thin layer of
ultrasound scanning gel (Clear Image Singles) was applied to
cover scanning area. Ultrasound co-registered PA imaging was
performed immediately using a 21 MHz transducer at 100%
power, 40 dB PA gain, and excitation wavelength of 774 nm
(Fig. 4d). PA images were processed using Image J software,
and PA intensity was quantified after subtraction of background
signals (Fig. 4e, 4f).
#S.R.K. is currently at the Department of Biomedical Engineering,
Pennsylvania State University.
‡Deceased on July 18, 2020.
Funding Sources
This work was supported by the Stanford University National
Cancer Institute (NCI) Centers of Cancer Nanotechnology
Excellence U54CA199075 (to JR) and NIGMS R35GM124963 (to
LC).
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Conflict of Interests
Dr. Gambhir served on the board of directors of Endra Inc. and as
a consultant for Visualsonics Inc.; both manufacturers of PA
imaging equipment for small animals.
Fluorescence imaging of polymerizable probe in tumor
models. To initiate tumors in female nu/nu mice (8-week-old,
Charles River Inc.), 5-million U87MG cells suspended in saline
(50 μL, original culturing medium was replaced immediately
before implantation) were injected subcutaneously at the left
shoulder of the mouse. Tumors were grown until reaching the
largest dimension at 0.5 to 0.7 cm (~14 to 21 days). The mice
were anesthetized, and the tumors of two mice groups (n=5)
were treated with intratumoral injections of GSH (20 mM, pH
7.4) and a third group received saline (50 μL). Probe 1 or 2 (20
nmol in 200 μL saline) was administered 5 minutes later to the
two mice groups with GSH treatment via tail vein, and probe 1
(20 nmol in 200 μL saline) was also administered to the third
mice group with saline treatment. Longitudinal whole animal
fluorescence was monitored over 24 hours using an IVIS
spectrum fluorescence imaging system (PerkinElmer, Fig. 5a,
Supporting Fig. S10). Tumor fluorescence radiance was
quantified by region of interest (ROI) measurement using IVIS
Living Image 4.2 software (Fig. 5b, 5c).
ACKNOWLEDGMENT
We thank Prof. Axel T. Brunger (Stanford Molecular & Cellular
Physiology) for use of the SEC-MALS instrument, Dr. Sean
O'Leary (Joseph Puglisi lab) and Dr. Kimberly Brewer (Brian Rutt
lab) for initial characterization of the probes, Mr. Ken Lau and Dr.
Mark Stolowitz (Proteomics Core Facility, Stanford Canary
Center) for MALDI-MS analysis, Drs. Adam J. Shuhendler (Rao
lab) and Lihong Bu (Zhen Cheng lab) at Stanford School of
Medicine for their kind assistance in animal work, Deju Ye (Rao
lab) and Leonid Pereyaslavets (Michael Levitt lab) for helpful
discussions.
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