RESEARCH ARTICLE
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Truly Tiny Acoustic Biomolecules for Ultrasound Imaging
and Therapy
Bill Ling, Bilge Gungoren, Yuxing Yao, Przemysław Dutka, Reid Vassallo, Rohit Nayak,
Cameron A. B. Smith, Justin Lee, Margaret B. Swift, and Mikhail G. Shapiro*
Nanotechnology offers significant advantages for medical imaging and
therapy, including enhanced contrast and precision targeting. However,
integrating these benefits into ultrasonography is challenging due to the size
and stability constraints of conventional bubble-based agents. Here bicones,
truly tiny acoustic contrast agents based on gas vesicles (GVs), a unique class
of air-filled protein nanostructures naturally produced in buoyant microbes,
are described. It is shown that these sub-80 nm particles can be effectively
detected both in vitro and in vivo, infiltrate tumors via leaky vasculature,
deliver potent mechanical effects through ultrasound-induced inertial
cavitation, and are easily engineered for molecular targeting, prolonged
circulation time, and payload conjugation.
1. Introduction
Ultrasound is a powerful modality for medical diagnostics and
treatment due to its noninvasive nature, real-time imaging
B.Ling,B.Gungoren,Y.Yao,P.Dutka,R.Nayak,C.A.B.Smith,
M. B. Swift, M. G. Shapiro
Division of Chemistry and Chemical Engineering
California Institute of Technology
Pasadena, CA 91125, USA
E-mail:
mikhail@caltech.edu
P.Dutka,J.Lee
DivisionofBiologyandBioengineering
CaliforniaInstituteofTechnology
Pasadena,CA91125,USA
R.Vassallo
SchoolofBiomedicalEngineering
UniversityofBritishColumbia
Vancouver,BC, V6T1K2,Canada
M.G.Shapiro
DivisionofEngineeringandAppliedScience
CaliforniaInstituteofTechnology
Pasadena,CA91125,USA
M.G.Shapiro
HowardHughesMedicalInstitute
CaliforniaInstituteofTechnology
Pasadena,CA91125,USA
The ORCID identification number(s) for the author(s) of this article
can be found under
https://doi.org/10.1002/adma.202307106
© 2024 The Authors. Advanced Materials published by Wiley-VCH
GmbH. This is an open access article under the terms of the
Creative
Commons Attribution
License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
DOI: 10.1002/adma.202307106
capabilities, and widespread accessibility.
[
1
]
Incorporation of nanomaterial probes
could significantly enhance these benefits,
enabling immune evasion, molecular tar-
geting, extravasation, and multifunctional
strategies for improved contrast and drug
delivery.
[
2,3
]
Although such probes have
proven successful in modalities such as nu-
clear and magnetic resonance imaging,
[
4
]
designing truly nanoscale agents for ul-
trasound continues to pose a challenge.
Conventional agents, formulated as lipid-
shelled gas microbubbles, are typically
limited by surface tension to sizes larger
than 1
μ
m,
[
5
]
while proposed submicron
agents based on bubbles,
[
6
]
droplets,
[
7
]
phase-change,
[
8,9
]
and gas-trapping
particles
[
10–12
]
remain relatively large (
>
200 nm) and can be diffi-
cult to prepare.
In this study, we introduce bicones, truly tiny acoustic re-
porters based on gas vesicles (GVs), a class of air-filled protein
nanostructures assembled by certain aquatic microbes for buoy-
ancy regulation.
[
13,14
]
GVs comprise a corrugated protein shell of
varying thickness (1–3 nm) that excludes liquid water while al-
lowing dynamic gas exchange, creating a stable nanoscale pocket
of air.
[
15,16
]
Acoustic waves strongly scatter at this air-liquid in-
terface, enabling GVs to serve as contrast agents
[
14,17
]
and re-
porter genes
[
18,19
]
for ultrasound imaging. GVs are highly versa-
tile, with physical and acoustic properties that are easily tuned
by functionalizing the protein shell or by modifying their con-
stituent genes.
[
20,21
]
Additionally, GVs can serve as therapeu-
tic platforms by stimulating ultrasound mechanotherapy
[
22
]
and
photodynamic therapy.
[
23
]
However, their largest dimensions typ-
ically exceed 200 nm, putting them on the larger end of biological
nanomaterials.
GV formation begins as a soluble nucleation complex, pro-
gresses into a biconical structure, and ultimately elongates into
a cylindrical shape.
[
15,16
]
We hypothesized that we could produce
ultrasmall particles by inhibiting GV growth at the bicone stage.
In this study, we test this idea and establish the fundamental
properties and capabilities of GV bicones in ultrasound imag-
ing and therapy, evaluating their imaging contrast, tumor uptake
capacity, and interactions with focused ultrasound (FUS). Addi-
tionally, we devise surface engineering strategies to extend circu-
lation time, target specific cells, and carry protein cargo. Using
these truly tiny acoustic biomolecules, we aim to enable further
integration of ultrasonography with nanomedicine.
Adv. Mater.
2024
, 2307106
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2. Results
2.1. Bicones are Truly Tiny Ultrasound Contrast Agents
We hypothesized that we could disrupt GV growth by deleting
gvpN
, a gene essential for GV elongation.
[
24,25
]
We modified a
previously described bacterial GV cluster containing structural
proteins from
Anabaena flos-aquae
and chaperones from
Bacillus
megaterium
[
18
]
and expressed this construct in
E. coli
(
Figure
1
a).
As hypothesized, transmission electron microscopy (TEM)
showed uniformly small, biconical particles (Figure
1b). Further
deletions of
gvpC
,
gvpR
,
gvpT
,and
gvpU
did not affect morphol-
ogy (Figure
S1, Supporting Information). Cryogenic electron mi-
croscopy (cryo-EM) showed that the bicone shell comprised two
low-pitch helices beginning at each tip and converging in the
center (Figure
1c), consistent with the structure of GVs.
[
16
]
In-
dividual particles had an average diameter of 39.7
±
0.5 nm and
length of 72.3
±
1.1 nm (Figure
1d). Bicones were colloidally sta-
ble in phosphate-buffered saline (PBS), with a hydrodynamic di-
ameter of 58.4
±
0.5 nm, as measured by dynamic light scat-
tering (DLS) (Figure
1e), and zeta potential of
−
34.9
±
1.2 mV
(Figure
1f). Hydrodynamic diameter increased slightly following
a 5-week incubation at 4
°
C, suggesting small amounts of aggre-
gation, though TEM showed that particles remained primarily
dispersed (Figure
S2, Supporting Information). Assuming a 2-
nm thick shell,
[
13,26
]
we estimated that each particle had a molec-
ular weight of 5.94 MDa and enclosed 0.023 aL of gas (76.3%
of total volume) (Figure
S3 and Table
S1, Supporting Informa-
tion). The volume of a typical bicone is 93 times smaller than the
average GV used in most ultrasound experiments and 17 times
smaller than a lentiviral capsid (Figure
1g; Table
S1, Supporting
Information).
Bicones are expected to be highly resistant to external pres-
sure, as shell robustness is inversely related to diameter.
[
27
]
We
tested this using pressurized absorbance spectroscopy, which
measures optical density (OD) under increasing hydrostatic pres-
sure to identify the threshold when GVs collapse and lose
their internal gas content and consequent ability to scatter
light.
[
28
]
OD decreased gradually between 0.9 and 1.3 MPa,
with a midpoint at 1.16 MPa (Figure
1h). To assess collapse
under acoustic pressure, we embedded bicones in an agarose
phantom and acquired B-mode ultrasound images at a cen-
ter frequency of 15 MHz, with increasing transducer driv-
ing voltage. Contrast diminished sharply at 2 MPa and was
completely erased at 3 MPa, with a midpoint of ̃2.4 MPa
(Figure
1i). These collapse thresholds rank bicones as the stur-
diest GV variant we have developed (Figure
S4, Supporting
Information).
Having confirmed that our transducers could collapse bi-
cones, we visualized them using BURST imaging.
[
29
]
This
method maximizes sensitivity and specificity for GVs by cap-
turing the transient signals generated during acoustic col-
lapse. BURST signal correlated linearly with concentration
and was reliably detected at 0.5 nM, the lowest concentra-
tion tested (Figure
1j,k). In comparison, concentrations be-
low 6.5 nM were difficult to visualize using B-mode imaging
(Figure
S5, Supporting Information). Contrast was preserved
after 5 weeks of incubation at 4
°
C(Figure
S2, Supporting
Information).
2.2. Bicones Enable Visualization of Tumors
We next investigated the ability of bicones to produce ultra-
sound contrast in vivo. We hypothesized that bicones could be
visualized in mouse xenografts, as sub-100 nm nanoparticles
are expected to accumulate in cancerous tissue through leaky
vasculature.
[
30–32
]
To test this hypothesis, we intravenously (IV)
administered 200
μ
L of an 84 nM bicone suspension (total dose
of 10.1 trillion particles or 100
μ
g protein) into nude mice bearing
subcutaneous U-87 MG tumors and performed BURST imaging
after 1 h. We chose this dose to maximize delivery by exploit-
ing potential clearance saturation mechanisms.
[
33
]
We expected
to see contrast within a several-millimeter band of depth due to
transducer focusing and the high collapse pressure of bicones.
Indeed, BURST signal was detected as a punctate band within
the tumor, which was absent during a subsequent acquisition,
confirming its specificity to intact particles (
Figure
2
a; Figure
S6,
Supporting Information). We validated tumor accumulation in a
separate group of mice by measuring the fluorescence of tumors
resected 2 h after injection of 12.6 pmol bicones (75
μ
g protein)
labeled with a near-infrared dye (Figure
2b,c).
To examine biodistribution, we acquired fluorescence images
of mouse organs excised at predetermined intervals following IV
injection of 16.8 pmol bicones (100
μ
g protein) labeled with a far-
red fluorescent dye (Figure
2d,e). Fluorescence was highest in the
liver, spleen, and kidneys. At 3 h post-injection, mean intensities
in these organs were 30.5, 10.7, and 11.0, respectively, decreas-
ing to 9.4, 3.9, and 4.1 after 24 h and further to 2.6, 1.2, and 1.7
after 48 h. This gradual reduction in fluorescence over time indi-
cates active elimination of bicones from the body, with the high
kidney signal suggesting lysosomal degradation followed by re-
nal excretion.
[
34,35
]
Fecal excretion may also contribute to elim-
ination, as shown by biliary and intestinal accumulation of GVs
from
H. salinarum
,
[
36
]
though we did not examine these organs in
this study. Compared to larger GVs,
[
34,36
]
bicones showed consid-
erably lower uptake in the spleen and lungs, consistent with the
ability of smaller particles to bypass filtration by these organs.
[
37
]
Overall, our data demonstrate that bicones are remarkably small
acoustic biomolecules that can be easily produced in bacteria and
detected both in vitro and in vivo.
2.3. Bicones can Seed Inertial Cavitation
Having demonstrated bicone accumulation in tumors, we next
explored their potential for therapeutic applications. Specifically,
we investigated their capacity to seed inertial cavitation under
FUS.
[
22
]
This phenomenon can be used to precisely disrupt tis-
sue, promote drug penetration, and eliminate diseased cells.
[
38
]
Inertial cavitation involves the growth of a bubble—often nu-
cleated by air carried in synthetic agents
[
11
]
or released during
GV collapse
[
22
]
—through coalescence across several acoustic cy-
cles, culminating in a high-energy implosion (
Figure
3
a). Bub-
bles undergoing this process generate distinct broadband acous-
tic emissions.
[
22
]
To test the ability of bicones to seed inertial cavitation, we in-
sonated samples with a 330 kHz FUS transducer and recorded
acoustic emissions using an orthogonally positioned imaging
transducer as a passive cavitation detector (PCD) (Figure
3b).
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Figure 1.
Bicones are truly nanoscale ultrasound contrast agents. a) Diagram of bicone structure and gene cluster. Gas can dynamically exchange
through the bicone shell, forming a thermodynamically stable pocket of air. Deletion of the
gvpN
gene prevents GVs from growing beyond the bicone
stage. b) Representative TEM images of bicones in
E. coli
(left, scale bar, 500 nm) and after purification (right, scale bar, 100 nm). c) Representative
cryo-EM image of purified bicones showing the helical shell structure. Scale bar, 25 nm. d) Distribution of lengths and diameters of individual partic
les,
measured manually from cryo-EM images.
N
=
100. e) Intensity-weighted DLS measurements of bicones in PBS.
N
=
35. Error bars,
±
SEM. f) Zeta
potential measurements.
N
=
6. Error bars,
±
SEM. g) Diagram comparing the dimensions of a typical microbubble, nanobubble, GV, lentivirus, bicone,
and adeno-associated virus (AAV). h) Pressurized absorbance spectroscopy measurements of bicones in PBS, normalized to starting OD.
N
=
6. Thick
line, mean; shaded area,
±
SEM. i) B-mode intensity of bicones embedded in an agarose phantom following exposure to pulses at the indicated acoustic
pressure, normalized to the starting value.
N
=
6. Thick line, mean; shaded area,
±
SEM. j) Representative BURST images of bicones embedded in an
agarose phantom at concentrations between 0 and 33.7 nM, overlaid on a B-mode image to show sample outlines. Scale bars, 1 mm. k) Quantification
of BURST signal within samples from panel J. Data were fit by linear regression, slope
=
0.86, r
2
=
0.5674.
N
=
12. Error bars,
±
SEM.
Adv. Mater.
2024
, 2307106
2307106 (3 of 11)
© 2024 The Authors. Advanced Materials published by Wiley-VCH GmbH
15214095, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202307106 by California Inst of Technology, Wiley Online Library on [27/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License