PNAS
2023 Vol. 120 No. 39 e2309822120
https://doi.org/10.1073/pnas.2309822120
1 of 5
RESEARCH ARTICLE
|
Significance
Remote control of chemical
reactions with spatial and
temporal precision would be an
enabling technology for diverse
biomedical applications. Polymer
mechanochemistry offers a
promising approach whereby
ultrasound is used to activate
specific chemical transformations.
However, ultrasound conditions
conventionally employed in the
field result in unsafe heating and
tissue damage. To address this
challenge, we have developed a
synergistic platform that leverages
air
-
filled protein nanostructures
(gas vesicles) to serve as acousto
-
mechanical transducers, enabling
mechanochemical activation
under clinically relevant conditions
using biocompatible focused
ultrasound for the controlled
release of small molecule
payloads, including a
chemotherapeutic drug. This
strategy represents a promising
modality for controlling chemical
reactivity in biological systems and
unlocks the translational potential
of polymer mechanochemistry for
therapeutic and bioimaging
applications.
This article is a PNAS Direct Submission.
Copyright © 2023 the Author(s). Published by PNAS. This
open access article is distributed under
Creative Commons
Attribution
-
NonCommercial
-
NoDerivatives License 4.0 (CC
BY
-
NC
-
ND)
.
1
Y.Y. and M.E.M. contributed equally to this work.
2
To whom correspondence may be addressed. Email:
mikhail@caltech.edu or mrobb@caltech.edu.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2309822120/-
/DCSupplemental
.
Published September 19, 2023.
CHEMISTRY
APPLIED BIOLOGICAL SCIENCES
Remote control of mechanochemical reactions under
physiological conditions using biocompatible focused
ultrasound
Yuxing Yao
a
,1
, Molly E. McFadden
a
,1
, Stella M. Luo
a
, Ross W. Barber
a
, Elin Kang
a
, Avinoam Bar
-
Zion
a
, Cameron A. B. Smith
a
, Zhiyang Jin
b
,
Mark Legendre
a
, Bill Ling
a
, Dina Malounda
a
, Andrea Torres
a
, Tiba Hamza
a
, Chelsea E. R. Edwards
a
, Mikhail G. Shapiro
a,b,c
,2
,
and Maxwell J. Robb
a
,2
Edited by Steve Granick, University of Massachusetts at Amherst, Amherst, MA; received June 15, 2023; accepted August 1, 2023
External control of chemical reactions in biological settings with spatial and temporal
precision is a grand challenge for noninvasive diagnostic and therapeutic applications.
While light is a conventional stimulus for remote chemical activation, its penetration is
severely attenuated in tissues, which limits biological applicability. On the other hand,
ultrasound is a biocompatible remote energy source that is highly penetrant and offers
a wide range of functional tunability. Coupling ultrasound to the activation of specific
chemical reactions under physiological conditions, however, remains a challenge. Here,
we describe a synergistic platform that couples the selective mechanochemical activation
of mechanophore
-
functionalized polymers with biocompatible focused ultrasound (FUS)
by leveraging pressure
-
sensitive gas vesicles (GVs) as acousto
-
mechanical transducers.
The power of this approach is illustrated through the mechanically triggered release of
covalently bound fluorogenic and therapeutic cargo molecules from polymers containing
a masked 2
-
furylcarbinol mechanophore. Molecular release occurs selectively in the
presence of GVs upon exposure to FUS under physiological conditions. These results
showcase the viability of this system for enabling remote control of specific mechano-
chemical reactions with spatiotemporal precision in biologically relevant settings and
demonstrate the translational potential of polymer mechanochemistry.
polymer mechanochemistry | focused ultrasound | drug delivery | mechanophore | gas vesicle
External control of specific chemical reactions with spatial and temporal precision is a grand
challenge for biomedical applications. For example, the targeted delivery of therapeutic
compounds to precise locations in the body maximizes accumulation of drugs at the sites
of disease and reduces undesirable side effects to healthy tissues (1–4). Likewise, spatially
triggered reactions can establish patterned engineered tissues and functional living materials
(5–8). Among various ways to drive chemical transformations remotely, light is a proto-
typical external stimulus for achieving high spatiotemporal resolution (9, 10), but its limited
tissue penetration depth hampers biological utility (11). Research in the nascent field of
polymer mechanochemistry has focused on the development of force
-
sensitive molecules
termed mechanophores (12), including those that release covalently bound small molecules
upon mechanochemical activation (13, 14). Mechanical force, which can also be applied
with spatial and temporal precision, is transduced to the mechanophore through covalently
linked polymer chains to elicit a chemoselective response. Mechanophores have been devel
-
oped that release carbon monoxide (15, 16), acid (17, 18), catalysts (19), and a variety of
other payloads (13). Research from the groups of Göstl and Herrmann (20, 21) and the
Robb group (22–24) has focused on the design of more general and modular mechanophore
platforms enabling the mechanically triggered release of functionally diverse molecules that
are especially promising for biomedical applications (13).
Ultrasonication is an effective technique for achieving mechanophore activation in the
laboratory. High
-
intensity low
-
frequency sonication (typically 20 kHz) using an immer-
sion probe is commonly employed to exert mechanical forces on polymers in solution via
acoustic cavitation, a process in which the nucleation, growth, and collapse of gas bubbles
produce solvodynamic shear and results in the rapid elongation of polymer chains (Fig.
1
A
)
(12, 25). However, the cavitation of dissolved gases under these conditions is highly
destructive to tissues, making it incompatible with most biological applications (13, 26).
Recent studies have begun to explore the use of clinically relevant focused ultrasound
(FUS) operating at higher frequencies for mechanophore activation in polymer networks
(27) and hydrogels (28). FUS is an ideal external stimulus as it can be applied with sub-
millimeter spatial resolution and is highly penetrant to biological tissues (29). Nevertheless,
while these earlier studies provide a promising direction, the high acoustic intensities
OPEN ACCESS
Downloaded from https://www.pnas.org by George Porter on September 21, 2023 from IP address 131.215.252.179.
2 of 5
https://doi.org/10.1073/pnas.2309822120
pnas.org
employed cause unsafe heating, which represents a major obstacle
for biomedical applications (27, 28, 30). Moreover, coupling bio
-
compatible ultrasound with the remote activation of covalent
mechanochemical reactions in solution remains an unsolved chal
-
lenge. New methodologies are therefore required to realize the
translational potential of polymer mechanochemistry (13).
Gas vesicles (GVs) are a unique class of genetically encodable,
air
-
filled protein nanostructures that have been developed as con
-
trast agents for noninvasive biomedical imaging, including
high
-
frequency diagnostic ultrasound (31–34). GVs typically have
diameters of
~
100 nm and lengths of
~
500 nm and comprise
amphiphilic protein shells that are gas
-
permeable but exclude liq
-
uid water from their hydrophobic interior (35). Recently, the
Shapiro group found that GVs act as seeds for bubble formation
and cavitation upon exposure to biocompatible FUS in
vivo (36).
Under sustained ultrasound pulses with relatively low pressure
levels, larger gas bubbles liberated from the rupture of GVs
undergo rapid growth followed by intense collapse in an inertial
cavitation process with pronounced mechanical effects. With the
ultrasound parameters used, these effects take place only in the
vicinity of GVs and are benign to other tissues (36). Based on
these results, we hypothesized that GVs could function as
acousto
-
mechanical transducers to effectively couple FUS oper-
ating under physiological conditions with the mechanochemical
activation of mechanophore
-
containing polymers (Fig. 1
B
).
Herein, we describe an approach to achieve remote control of
mechanochemical reactions in aqueous environments using bio-
compatible FUS. Using GVs as acousto
-
mechanical transducers,
FUS selectively triggers the release of fluorogenic and therapeutic
cargo molecules from mechanophore
-
functionalized polymers.
This methodology represents a promising step forward for bridg-
ing the gap between the technological potential of polymer mech
-
anochemistry and translational applications in biology, medicine,
and living materials.
Results and Discussion
We initially set out to identify FUS parameters for safe operation
under physiological conditions with a particular emphasis on
maintaining low
-
to
-
moderate pressure levels and avoiding tem-
perature increases greater than
~
6 °C to stay within a biomedically
relevant regime. FUS applied at high acoustic intensities can
induce coagulative necrosis in tissues resulting from thermal dis-
ruption (37). We performed experiments with 330 kHz FUS on
either an 800
-
μL water sample in a sealed plastic microcentrifuge
tube or a tissue
-
mimicking agarose gel inside a water tank and
monitored peak negative pressure (PNP) and sample temperature
using a hydrophone and internal thermocouple, respectively
(Fig. 2
A
). An upper limit for PNP of 1.47 MPa was established
with a 4.5% duty cycle (3,000 cycles per pulse with 5 Hz pulse
repetition frequency), which resulted in a maximum temperature
increase of only 3.6 °C in the agarose phantom (7.3 °C inside the
plastic tube) that equilibrated in <2 min (Fig.
2
B
). This biocom-
patible upper limit results in a spatial peak
-
temporal average
acoustic intensity (I
spta
) of 3.6 W/cm
2
that is within the safety
limits established for the use of ultrasound in therapeutic appli-
cations (38). Similar ultrasound parameters have been successfully
applied for therapeutic purposes to delicate tissues like the brain
in both live animal studies and human clinical trials (39, 40). We
note that using similar FUS parameters as those previously
employed for mechanophore activation (27, 28), at both 330 and
916 kHz, resulted in an unsafe temperature increase exceeding 27
°C above baseline due to the significantly higher acoustic intensity
(Fig. 2
B
and
SI Appendix
, Fig. S1
).
Having identified biocompatible FUS parameters, we next sought
to determine whether mechanophore activation could be achieved
under physiologically relevant conditions. We employed a masked
2
-
furylcarbinol mechanophore developed in the Robb group for
mechanically triggered molecular release (Fig.
2
C
) (22, 23). Briefly,
mechanochemical activation of the furan–maleimide Diels–Alder
Fig. 1.
Activation of mechanochemical reactions under physiological
conditions using biocompatible FUS enabled by gas vesicles (GVs) as acousto
-
mechanical transducers. (
A
) Conventional methods of achieving ultrasound
-
induced mechanochemical activation rely on the cavitation of dissolved gases
in solution using hazardous sonication at low frequency and high acoustic
intensity. (
B
) Using GVs, which function as acousto
-
mechanical transducers,
mechanochemical activation of mechanophore
-
functionalized polymers is
achieved using biocompatible FUS under physiological conditions, enabling
the triggered release of molecular payloads.
05
01
00
150
0
10
20
30
40
T(
C)
Ti
me
(s)
Protic
Media
Cargo
Me
N
O
O
O
H
H
Polymer
Polymer
OPh
Functional
Cargo
Masked 2-Furylcarbinol
Mechanophore
O
Cargo
Me
Polymer
OPh
Mechanical
Force
C
2.2
2.4
3.6
4.8
4.0
5.7
6.5
9.8
7.7
9.4
12
27
4.5
9.1
18.2
1.17
1.31
1.47
1.85
Duty Cycle (%)
Max.
T (
o
C)
Peak
Negative
Pressure,
PNP (MPa)
5
10
15
20
25
A
B
1.9M
Pa
,40W
/cm
2
(tube)
1.47
MP
a, 3.6W
/cm
2
(tube)
1.47
MP
a, 3.6W
/cm
2
(agarose
)
Fig. 2.
Analysis of biocompatible FUS conditions for mechanochemical
activation. (
A
) Equilibrated maximum temperature increment (max.
Δ
T) at
the focus of ultrasound in a tissue
-
mimicking 1% agarose hydrogel phantom
with varying PNP and duty cycle. (
B
) Temperature profile of an 800
- μ
L water
sample at the focus of ultrasound inside a 2
-
mL plastic microcentrifuge tube or
a tissue
-
mimicking agarose gel (phantom) with different ultrasound conditions
(1.47 MPa PNP, 330 kHz, pulsed wave, 3,000 cycles each pulse, 4.5% duty
cycle, 3.6 W/cm
2
acoustic intensity, or 1.9 MPa PNP, 330 kHz, continuous wave,
10 s on/20 s off, 4 repeats, and 40 W/cm
2
acoustic intensity). (
C
) Reaction
scheme illustrating mechanically triggered molecular release from a masked
2
-
furylcarbinol mechanophore incorporated near the center of a polymer.
Downloaded from https://www.pnas.org by George Porter on September 21, 2023 from IP address 131.215.252.179.
PNAS
2023 Vol. 120 No. 39 e2309822120
https://doi.org/10.1073/pnas.2309822120
3 of 5
adduct reveals a latent 2
-
furylcarbinol derivative that spontaneously
decomposes to release a covalently bound cargo molecule. Release
occurs efficiently in polar protic environments, and the molecular
design is amenable to payloads conjugated to the mechanophore via
a range of common functional groups including alcohols and amines
(23). The incorporation of cargo molecules through covalent linkages
increases resistance to nonspecific payload release in the absence of
a specific triggering event compared to noncovalent encapsulation
strategies (41), while the ability to precisely modulate the reactivity
of mechanophores through structural modification using the tools
of organic chemistry affords excellent selectivity and control over
molecular release (13). We further identified the water
-
soluble, non
-
toxic polymer poly[2
-
(methylsulfinyl)ethyl acrylate] (PMSEA) (42)
that can be synthesized using controlled radical polymerization,
enabling straightforward incorporation of the mechanophore near
the middle of the polymer chain where force is maximized during
solvodynamic extension (12). We note that alternative polymer con
-
jugation strategies are also readily available, such as “click” coupling
(43, 44), to further broaden the scope of accessible materials.
We first investigated a PMSEA polymer containing a chain
-
centered mechanophore loaded with a fluorogenic aminocoumarin
payload (
PMSEA
-
CoumNH
2
,
M
n
= 260 kg/mol;
Ð
= 1.47)
(Fig. 3
A
and see
SI Appendix
for details) (23). Release of the ami-
nocoumarin small molecule results in a pronounced increase in
fluorescence that is easily detected and quantified spectroscopically.
Exposing a dilute solution of the polymer (2 mg/mL) in water to
330 kHz FUS under the conditions identified above in the pres-
ence of GVs (1.4 nM) results in a strong fluorogenic response
indicating the successful release of aminocoumarin (Fig.
3
A
and
B
). Approximately 15% release was observed after 10 min of FUS
exposure (Fig. 3
C
). Given their relative concentrations,
~
800
equivalents of
PMSEA
-
CoumNH
2
were activated per GV within
this timeframe. Extended exposure to FUS resulted in additional
aminocoumarin release (
SI Appendix
, Fig. S2
). Importantly, a
fluorogenic response was not observed in the absence of GVs under
the same conditions, confirming the GVs function as essential
acousto
-
mechanical transducers, enabling mechanophore activa-
tion and selective cargo release under biocompatible conditions.
Additional control experiments were performed on an analogous
polymer with the mechanophore located at the chain end, which
is not subjected to mechanical force (12). Minimal aminocoumarin
release was observed under otherwise identical experimental con-
ditions, confirming the mechanochemical origin of molecular
release from
PMSEA
-
CoumNH
2
upon exposure to FUS in the
presence of GVs (Fig. 3
B
and
C
and
SI Appendix
, Fig. S3
).
Next, we set out to investigate the mechanically triggered
release of a small molecule therapeutic to demonstrate the capa
-
bility of this system for future drug delivery applications. We
selected camptothecin as a model chemotherapeutic anticancer
agent (45). Following a similar procedure as above, camptoth-
ecin was conjugated to the mechanophore bis
-
initiator through
a carbonate linkage followed by polymerization to afford
PMSEA
-
CPT
(
M
n
= 319 kg/mol;
Ð
= 1.47) (Fig.
4
A
and see
SI Appendix
for details). Preliminary experiments performed on
an isolated small molecule furfuryl carbonate model compound
resembling the intermediate that is unmasked upon mechano-
phore activation confirmed that camptothecin is successfully
released in aqueous media, as evidenced by
1
H NMR spectros-
copy and high
-
performance liquid chromatography (HPLC)
measurements (
SI Appendix
, Figs.
S4 and S5
). Importantly,
PMSEA
-
CPT
is stable in aqueous solution, with negligible
release of camptothecin detected over a period of 2 mo under
ambient conditions (
SI Appendix
, Fig. S6
).
The triggered release of camptothecin from
PMSEA
-
CPT
was
then investigated using biocompatible FUS in the presence of GVs
under the physiologically relevant conditions established above
(Fig. 4
A
). Approximately 8% release of camptothecin was achieved
after 10 min exposure to FUS in the presence of GVs, as evidenced
by quantitative HPLC measurements (Fig.
4
B
). Similar to the
results above for the release of aminocoumarin, no camptothecin
release was detected upon exposure of
PMSEA
-
CPT
to FUS in
the absence of GVs. To assess the feasibility of this synergistic plat-
form for targeted chemotherapy, cytotoxicity was investigated
in
vitro on the viability of Raji cells, a diffuse large B cell lymphoma
model of non
-
Hodgkin lymphomas, using an MTT colorimetric
assay (46). Cells were treated with samples of
PMSEA
-
CPT
before
and after mechanochemical activation with FUS at various con-
centrations and incubated for 2 d at 37 °C (Fig.
4
C
). Cells incu-
bated with
PMSEA
-
CPT
that was previously exposed to FUS in
the presence of GVs exhibited a significant decrease in viability
with a half
-
maximal effective concentration (EC
50
) of
~
250 nM.
This EC
50
is approximately one order of magnitude higher than
cells treated with isolated camptothecin small molecule, fully con-
sistent with the extent of cargo release after 10 min of insonation
characterized above. In contrast, no significant cytotoxicity was
observed across all polymer concentrations in control experiments
performed with
PMSEA
-
CPT
that was not exposed to FUS, with
PMSEA
-
CPT
that was exposed to FUS in the absence of GVs, or
with a chain
-
end functional control polymer (
PMSEA
-
Control
)
subjected to identical FUS conditions in the presence of GVs.
Fig. 3.
Mechanochemically triggered release of an aminocoumarin small
molecule payload using biocompatible FUS. (
A
) Biocompatible FUS (330 kHz,
1.47 MPa PNP, 3.6 W/cm
2
, 3,000 cycles, 4.5% duty cycle, 10 min) triggers
the release of aminocoumarin from
PMSEA
-
CoumNH
2
(2 mg/mL in water)
selectively in the presence of GVs (1.4 nM) resulting in a fluorogenic response.
(
B
and
C
) Release of aminocoumarin characterized by photoluminescence
(PL) spectroscopy. Error bars represent SD from three replicate experiments
for (−GV −FUS) and (−GV +FUS) and from four replicate experiments for (+GV
+FUS). Asterisks represent statistical significance by ordinary one
-
way ANOVA
with multiple comparison (****
P
< 0.0001; ***
P
< 0.001; **
P
< 0.01; and ns,
not significant).
Downloaded from https://www.pnas.org by George Porter on September 21, 2023 from IP address 131.215.252.179.