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Current Medicinal Chemistry, XXXX, XX, XX-XX 1

REVIEW ARTICLE

Biogenic Gas Vesicles for Ultrasound Imaging and Targeted Thera-

peutics
Rui Wang1,2, Lufang Wang1,2, Yihan Chen1,2, Yuji Xie1,2, Mengrong He1,2, Ye Zhu1,2,
Lingling Xu1,2, Zhengyang Han1,2, Dandan Chen1,2, Qiaofeng Jin1,2,*, Li Zhang1,2,* and
Mingxing Xie1,2,*
1
Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China; 2Hubei Province Key Laboratory of Molecular Imaging, Wuhan, China

ARTICLE HISTORY
Received: February 11, 2021
Revised: May 01, 2021
Accepted: May 15, 2021
DOI:
10.2174/0929867328666210705145642
Keywords: Ultrasound imaging, gas vesicles, acoustic reporter gene, genetic engineering, cavitation, drug delivery.
Abstract: Ultrasound is not only the most widely used medical imaging mode for diag-
nostics owing to its real-time, non-radiation, portable and low-cost merits, but also a
promising targeted drug/gene delivery technique by producing a series of powerful bioef-
fects. The development of micron-sized or nanometer-sized ultrasound agents or delivery
carriers further makes ultrasound a distinctive modality in accurate diagnosis and effec-
tive treatment. In this review, we introduce one kind of unique biogenic gas-filled protein
nanostructures called gas vesicles, which present some unique characteristics beyond the
conventional microbubbles. Gas vesicles can not only serve as ultrasound contrast agent
with innovative imaging methods such as cross-amplitude modulation harmonic imaging,
but also can further be adjusted and optimized via genetic engineered techniques. Moreo-
ver, they could not only serve as acoustic gene reporters, acoustic biosensors to monitor
the cell metabolism, but also serve as cavitation nuclei and drug carrier for therapeutic
purpose. We focus on the latest development and applications in the area of ultrasound
imaging and targeted therapeutics, and also give a brief introduction to the corresponding
mechanisms. In summary, these biogenic gas vesicles show some advantages over con-
ventional MBs that deserve making more efforts to promote their development.

1. INTRODUCTION applications in the ultrasound area. Gas is encapsulated

Among many imaging technologies, ultrasound is
one of the most widely used diagnostic methods in
modern clinical medicine owing to its real-time, non-
radiation, portable and low-cost merits. In recent years,
with the in-depth understanding of the biological effect
of ultrasound, ultrasound medicine is developing to-
wards the direction of early detection, accurate diagno-
sis and multi-functional integration of diagnosis and
treatment [1]. At the same time, a large number of mi-
cron-sized or nanometer-sized ultrasound agents, such
as nanoparticles [2], microbubbles [3], nanoemulsions
[4] and vesicles [5] are being developed for healthcare
*Address correspondence to these authors at the Department of
ultrasound, Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Tel/Fax: +86-27-85726386;
E-mails: xiemx@hust.edu.cn; jin_qf@hust.edu.cn;
zli429@hust.edu.cn
with protein, lipid, surfactant or polymer molecules
aiming at the dual purpose of early accurate diagnosis
and precise therapy of diseases as the contrast agents
for ultrasound imaging or the delivery vehicles for
therapeutic agents [6]. Here, we introduce a unique
class of gas-filled protein nanostructures called gas
vesicles (GVs), which are expressed intracellularly in
some bacteria and archaea. These gas-permeable vesi-
cles have the native function to regulate cell buoyancy
in aqueous environments [7, 8]. In recent years, how-
ever, they were discovered to have additional applica-
tions in biotechnology and medicine, and a lot of excel-
lent studies have shown interesting and amazing re-
sults; it’s, therefore, worth conducting a thorough re-
view of the GVs. Firstly, we start with a brief primer of
the background of GVs, summarizing their history,
basic genetic, structural, physical, and biochemical
properties. Secondly, we describe their acoustic behav-

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2 Current Medicinal Chemistry, XXXX, Vol. XX, No. XX
iors and development in ultrasound imaging. Thirdly,
we give an overview of the ongoing research on the
therapeutic application of GVs and their underlying
mechanisms.
2. BACKGROUND OF GVS
2.1. The Discovery of GVs
Initially observed in 1895 [9], GVs were first dis-
covered as an intracellular part with the native function
of regulating cellular buoyancy in order to maintain a
suitable depth for optimal access to light and nutrients
in the aqueous environments. In 1965 [10], GVs were
identified as components of gas vacuoles first found in
cyanobacteria, then GVs of different shapes and sizes
were also discovered in a range of heterotrophic bacte-
ria such as Psychromonas ingrahamii [11], Serratia sp.
ATCC 39006 [12], Bacillus megaterium [13] and Strep-
tomyces sp. CB03234-S [14], and also in some archaea
such as Halobacterium salinarum [15], Haloferax med-
iterranei [16] and Haloquadratum walsbyi [17]. In re-
cent years, their basic genetic, structural, biochemical,
and physical properties have been well-studied by pio-
neering biology groups [18-20].
2.2. Structure and Properties of GVs
GVs are spindle- or cylindrical- shaped gas-filled in-
tracellular organelles with a width of 45-200 nm and
length of 100-2000 nm [8] (Figs. 1a, b). Their wall is
gas-permeable and composed of a single layer of a 7-8
kDa hydrophobic protein, called gas vesicle protein A
(GvpA), which serves as the core protein that can ex-
clude water but allows gas to freely permeate in and out
of their hollow interior as gas wall [21]. GVs are found
permeable to oxygen, hydrogen, nitrogen, carbon diox-
ide, carbon monoxide and even to perfluorocyclobu-
tane(C4F8), which has a molecular collision diameter of
0.63 nm, suggesting that the wall has pores [8]. Besides,
there is a native outer scaffolding protein, secondary gas
vesicle protein C (GvpC), that strengthens the structure
of GVs by forming a mesh around the exterior surface of
GVs (Fig. 1c). In addition to GvpA and GvpC, a bunch
of 6-12 other genes is also needed to enable GV produc-
tion, encoding structural proteins and assembly factors
such as chaperones and nucleators [22]. Some crystal
structures of the gas vesicle protein had already been
determined in recent years (Fig. 1d) [23].
GVs are a masterpiece for ultrasound imaging. On
the one hand, their gas-contained structure enables
them to scatter sound waves and produce ultrasound
contrast, which shows their potential in ultrasound im-
aging. GVs can keep a perfect balance with their sur-
roundings gas dissolved in surrounding media to equil-
Wang et al.
ibrate with the gas in GVs on a microsecond timescale,
resulting in physically stable and rigid characters. This
remarkable characteristic makes GVs become promis-
ing and stable ultrasound contrast agents. On the other
hand, GVs collapse when the net pressure across the
gas vesicle wall exceeds the ‘critical collapse pressure’
[8]. This feature of GVs can be used to generate sub-
traction imaging, whereby images can be taken before
and after GVs collapse to allow a better contrast.
Moreover, GVs isolated from different species possess
detectable differences in acoustic properties, suggesting
a potential multiplexed imaging strategy [15]. Last but
not least, the mechanical and surface properties of GVs
can be engineered genetically by exchanging the native
outer GvpC with its recombinant variants. Therefore,
GVs can be modified and functionalized through genet-
ic and biochemical methods and be used not only in the
area of ultrasound contrast imaging but also in ultra-
sound targeted gene/drug delivery. Over the years, sci-
entists and researchers have been making efforts to
gain a deeper understanding of the acoustic properties
of GVs and the genetic engineering method of GVs
[24-27], their goals are to obtain nanostructures with a
new mechanical, surface, acoustic, and functional
properties to enable harmonic, multiplexed, and multi-
modal ultrasound imaging as well as localized or tar-
geted therapeutics.
3. GVS FOR ULTRASOUND IMAGING
Current ultrasound contrast agents are conventional-
ly gas microbubbles stabilized with lipid or protein
shells [28]. They have limitations due to their relatively
broad size distribution and inherent physical instability,
and Laplace pressure may further make their gas con-
tent gradually diffuse into the surrounding medium and
finally fall into fragmentation [29-31]. Besides, the mi-
cron size of bubbles makes them challenging to get
across the vascular barrier, thus limiting the usefulness
of these contrast agents when imaging vascular struc-
tures. Recently, GVs were found useful in a whole new
medical field - a novel contrast agent for ultrasound,
paving the way for more precise and direct visualiza-
tion of tissues, cells and even molecular functions. In
this section, we would introduce the application of GVs
as ultrasound contrast.
3.1. Development of GVs as an Ultrasound Contrast
Agent
In March 2014, Shapiro and his team proposed for
the first time that GVs can serve as a kind of ultrasonic
molecular reporter, producing ultrasound contrast both
in vivo and in vitro [28]. In their research, GVs
Biogenic Gas Vesicles for Ultrasound Imaging and Targeted Therapeutics Current Medicinal Chemistry, XXXX, Vol. XX, No. XX 3

Fig. (1). Structure and properties of GVs. (a). Resuspended milky white solutions of purified Ana (left), Halo (middle) and
Mega (right) GVs in PBS at OD500,ps ~6; (b). Representative TEM images for purified Halo, Ana and Mega GVs. Scale bars,
200 nm; (c). GvpA and GvpC are the two major structural constituents of GVs, with GvpA ribs (gray) forming the primary GV
shell and the outer scaffold protein GvpC (blue) conferring structural integrity. Each GvpC molecule has five 33-amino acid
repeats flanked by N- and C-terminal regions; (d). Structural superposition of Anabaena sp. PCC 7120GvpF (blue) on M. aeru-
ginosa PCC 7806 GvpF (orange; PDB ID: 4QSG). Reproduced with permission from ref. 20, 23 and 34. (A higher resolu-
tion/colour version of this figure is available in the electronic copy of the article).

generated stable signals and could be collapsed under a
specific hydrostatic pressure. Compared to buffer con-
trols, GVs prepared from both Anabaena flos-aquae
(Ana) and Halobacterium salinarum NRC-1 (Halo)
organisms can produce a strong ultrasound contrast for
up to a week at GV suspensions with an optical density
between 0.25 and 2.0. However, their strong contrast
disappeared after being collapsed by increasing acous-
tic pressure, confirming the researchers’ point of view.
This path breaking discovery opened the door to the
GVs development as reporters for the ultrasound and
other imaging tools, as well as novel nanoscale contrast
agents that can be functionalized and targeted for mo-
lecular imaging. Meanwhile, it is worth noting that the
critical acoustic collapse pressure of the two GVs is not
the same [32] (70-150 kPa for Halo and 440-605 kPa
for Ana). In addition, Halo GVs also have the charac-
teristics to generate nonlinear harmonic imaging and
improve the contrast to tissue ratio of ultrasound imag-
ing. Both second- and third-harmonic signals can be
observed at 12 MHz and 18 MHz for Halo GVs after
signal processing with corresponding bandpass filters;
the images of Halo vesicles showed 3.7- and 4.6- times
stronger ultrasound contrast, respectively. Moreover,
the harmonic signals also disappeared after the collapse
of the GVs. In contrast, Ana GVs did not generate har-
monic signals at 6 MHz because the hydrostatic col-
lapse threshold of the Ana GVs was higher than that of
the Halo GVs. Subtraction images can be obtained due
to this feature and the negative effect of background
scattering on the contrast specificity can be reduced
[28]. Up to now, there are three types of GVs mainly
used for ultrasound imaging (Table 1).
3.2. The Mechanism of the Harmonic Signal Gener-
ated by GVs
Further, in order to gain a better understanding of
the mechanism of the harmonic signal generated by
GVs, researchers investigated the acoustic collapse
pressure and behavior of Halo GVs at center frequen-
cies ranging from 12.5 to 27.5 MHz, and found that
GVs collapsed when exposed to a 6-cycle 20 MHz ul-
trasound pulse at positive peak pressure over 576 KPa,
which is approximately 9 times higher than the critical
collapse pressure observed in quasi-static conditions.
This situation occurs due to the difference in the appli-
cation rate of the compressive forces. After applying
hydrostatic pressure to GVs, an equilibrium time of
approximately 7 seconds allows gas to flow out of
GVs, while for acoustic pressure, the equilibrium peri-
od allowing the gas to flow out may even be shorter
than the estimated 1.5 µs [33]. The gas flow is restrict-
ed by the compressed gas and shells resist pressure
[34]. A 2011 study showed that the rate of compression
affects the degree of deformation of a spherical shell or
other shaped shells, thereby changing the threshold for
shell collapse. In addition, the viscoelastic properties of
the surrounding medium also have a specific effect
4 Current Medicinal Chemistry, XXXX, Vol. XX, No. XX
Table 1. GVs with their ultrasonic characteristics.
Types of GVs
GVs -forming
mainly used for Shape
microorganisms
UCAs
Cylindrical-
Anabaena flos- shaped with
Ana
aquae conical tips [23,
34]
Halobacterium Spindle-shaped
halo
salinarum [7, 27]
Cylindrical-
Bacillus megateri-
mega shaped with
um
conical tips [13]
UCAs, ultrasound contrast agents.
[35]. In this research, the Halo GVs in a medium with a
viscosity of 0.89-8 mPa.s. behaved nonlinearly under
ultrasound at an incident pressure varying from160 kPa
to the collapse pressure, resulting in a second harmonic
intensity with an amplitude 2-6 dB lower than the fun-
damental wave. Furthermore, the Rayleigh-Plesset
model was used for buckling simulation and showed
that buckling is the mechanism of harmonic generation.
Dynamic finite element analysis attributes these results
to non-spherical geometric structures, confirming that
acoustic buckling pressure is the critical collapse pres-
sure under hydrostatic condition [33]. Such experi-
ments were also carried out using GVs purified from
Ana, and similar results were obtained either in their
native form or with the GvpC shell removed, which can
create a better harmonic ultrasound signal [34, 36].
3.3. Ultrasound Imaging Modes for GVs
Although the phenomenon and relevant mechanism
of harmonic ultrasound signal generation by GVs were
well investigated, imaging approaches taking ad-
vantage of this nonlinear behavior have not been fully
developed yet. In 2017, Maresca and his research team
combined finite element mechanical modeling with
experiments both in vitro and in vivo, aiming to estab-
lish a nonlinear relationship between given pressure
and backscatter at a fixed frequency [36]. According to
the above established relationship, amplitude modula-
tion (AM) sequence and pulse inversion (PI) sequence
were applied to GVs specific imaging. The obtained
images were compared on the contrast-to-noise ratio
(CNR) and contrast-to-artifact ratio (CAR) indicators,
and it was found that although the CNR of the two se-
quences were similar, AM had a higher contrast speci-
ficity, making it a better imaging strategy. However,
Wang et al.
Mean collapse
hydrostatic
Hydrodynamic Ultrasound Nonlinear ultra-
pressures
diameters (nm) contrast sound contrast
(kPa)
260-320
PDI (0.15-0.21) 587 [20] High [28] Yes [34]
[20]
240-340
PDI (0.17-0.26) 59 [20] Medium [28] Yes [33]
[20]
200-380
750 [20] Low [20] No [20]
PDI(0.23-0.34) [20]
the problem with this imaging strategy is that enhanced
artifacts will appear below the rich areas of GVs. To
solve this problem, the team proposed a cross-
amplitude modulation (xAM) imaging mode. The xAM
mode derives from counterpropagating wave interac-
tion theory and relies on the cross-propagation plane-
wave transmission of X-waves with a narrow aperture
to achieve artifact-free in vivo imaging. In both simula-
tions and experiments, it was found that the residual
xAM nonlinearity due to wave propagation decreases
with the increase of plane-wave cross-propagation an-
gle. Next, it also proved in the tissue phantom that the
imaging artifacts at the far end of the GVs enrichment
zone decreased and nearly disappeared completely at
angles above 16.5 degrees. Finally, xAM is capable of
highly specific in vivo imaging of GVs located in the
gastrointestinal tract, which paves the way for the visu-
alization of GVs as acoustic reporter genes. In addition,
the coherent synthesis of xAM data collected at four
different angles θ has also proved to be an effective
way to improve further CTR (contrast-to-tissue ratio)
and CAR [37].
3.4. Genetic Engineered GVs
Natural GVs have many advantages. For example,
they can produce stable ultrasound contrast. Compared
with microbubbles, they are smaller in size and easier
to prepare, and are therefore expected to be used for
extravascular imaging [28, 38]. However, like mi-
crobubble, GVs also have the problem of being cleared
by the reticuloendothelial system in practical applica-
tions. Fortunately, the genetic encodability and of GVs
increases the possibility of changing the acoustic prop-
erties of these contrast agents at their DNA sequence,
making it possible to transform natural GVs into a kind
Biogenic Gas Vesicles for Ultrasound Imaging and Targeted Therapeutics
of new nanosized bubbles with a different surface,
functional, acoustic and targeting properties. This
transformation, on the one hand, enables harmonic im-
aging of GVs that were not originally capable and
makes multiplexing imaging, multimodal imaging, etc.
possible [33]; on the other hand, it also makes GVs
promising as an acoustic reporter gene used in prokar-
yotic and even eukaryotic cells [39, 40].
Lakshmanan and his colleagues succeed in the har-
monic imaging , multiplexing and multimodal imaging
of GVs through genetically engineered Ana GVs [34].
Ana GVs are encoded by nine different gene clusters.
Studies showed that removing GvpC or shortening its
sequence can reduce the hydrostatic collapse threshold
of Ana GVs [41, 42]. Besides, GvpC can be fused with
bacterial or viral polypeptides. Therefore, GvpC can be
used as a platform of multi-purpose for transforming
GVs. Three variants of Ana GVs with different mechan-
ical properties were designed: ∆GvpC, which removed
the GvpC protein; ∆N&C, whose GvpC protein was
truncated, lacking N-terminal and C-terminal; GvpCWT,
whose GvpC protein is replaced with an engineered pro-
tein similar to the wild-type GvpC protein sequence. A
sequence of results demonstrated that the transformation
of GvpC could realize harmonic imaging, multiplexing
imaging and multimodal imaging. First, in the in vitro
experiment, compared with GvpCWT, the harmonic
signal of ΔGvpC was 3.71 times higher than that of
GvpCWT. Then ΔGvpC and GvpCWT were injected
into anesthetized live mice, and the results were con-
sistent with the in vitro experiments, which shows that
protein engineering can indeed change the acoustic
properties of nanostructures and achieve harmonic imag-
ing. Second, the critical acoustic collapse pressures of
three Ana GVs variants are different. Multiplexed imag-
es of these three GVs were obtained by pressure spec-
trum unmixing to achieve multiplexing imaging, which
shows that the combination of engineered GVs and pres-
sure spectrum unmixing can be applied to the ultrasound
imaging of multiple target molecules in the same sample
(Figs. 2a, b). Third, GvpC was fused with SpyTag (ST)
and reacted with the recombinantly expressed fluores-
cent protein SpyCatcher-mNeonGreen (SC-MNG), and
SDS-PAGE analysis confirmed the formation of
SpyTag-Spycatcher covalent bonds (Figs. 2c, d). The
ultrasound and fluorescence bimodal imaging of MNG-
labeled GVs were successfully obtained, indicating that
multimodal imaging can be achieved by engineering
modification of GVs [34]. In summary, this molecular
engineering capability will lead to a better way to obtain
hybrid or mutant GVs with better harmonic responses,
multiplexing, multimodal detection, and molecular tar-
Current Medicinal Chemistry, XXXX, Vol. XX, No. XX 5
geting, ensuring ultrasound is a high-performance mo-
dality for molecular imaging with GVs.
4. GVS AS ACOUSTIC REPORTER
In addition to genetic engineering of GVs, the dis-
covery of GVs as acoustic biomolecules also fulfills the
potential for their development as acoustic reporter
genes. For many years, researchers have been paying
efforts in imaging specific cells in tissues ranging from
brain to tumors, and molecules as well as their interac-
tions in space and time. Up to now, most common
methods for imaging, such as cellular progress rely
much on luminescent or fluorescent proteins. Among
those proteins, reporters that can be genetically encod-
ed, such as the green fluorescent protein (GFP), have
become one of the most powerful techniques for mo-
lecular imaging in tissues and cells [43]. However,
GFP and its derivatives have some limitations in intact
animals due to light scattering [44]. Besides, all these
luminescent of fluorescent proteins rely on oxygen to
activate fluorescence, thus making it impossible to be
used in the anaerobic situation [45-47]. Therefore, it is
an urgent requirement to search for a new kind of re-
porter gene that can image specific molecules and cel-
lular interactions in living bodies with excellent spatial
and temporal resolution.
4.1. GVs Gene Expression in Prokaryotic Cells
Shapiro and his research team proved that GVs
could be used as an acoustic reporter gene, expressed in
prokaryotic cells, achieving cell positioning and cell
function imaging, performing roles in ultrasound what
GFP and similar proteins have in optical imaging. In
2018, in a letter published on Nature, the GvpA and
GvpC genes from Anabaena algae were combined with
gvpR-gvpU from Bacillus megaterium to form ARG1,
which was then successfully expressed in E. coli and
could generate strong ultrasound contrast. Furthermore,
the expression of ARG1 can be regulated by a promot-
er controlled by the chemical inducer IPTG. Neither
the expression of ARG1 nor the ultrasonic exposure
caused any harmful effects, and the multiplexing imag-
ing can also be achieved by engineering the GvpC of
ARG1. Next, The E. coli strain Nissle1917 (ECN) cells
expressing ARG1 or lux were injected into the colon of
anesthetized mice. Ultrasound images clearly showed
the location of ECN cells in the colon, while optical
imaging can only see a certain location of ECN cells in
the abdomen of mice, proving the advantages of ultra-
sound in spatial positioning in deep organs over optical
imaging. This research proved the concept of GVs as
acoustic gene reporters with high spatial and temporal
6 Current Medicinal Chemistry, XXXX, Vol. XX, No. XX Wang et al.

Fig. (2). GVs for ultrasound imaging. (a). Ultrasound images of an agarose phantom containing wells with ΔGvpC, ΔN&C,
GvpCWT and a mixture of the three variants (all GVs at final OD 1.0 in PBS), acquired at 6.25 MHz. I0, before collapse; I1,
after collapse at 630 kPa I2: after collapse at 790 kPa I3: after collapse at 1230 kPa; (b). Spectrally unmixed images processed
from the raw ultrasound data in (a). The bottom panel shows an overlay of the three unmixed channels C1, C2, and C3; (c). Top
panel: Ultrasound images of engineered and SC-mNG reacted GVs at OD 2.5 in PBS, acquired using a 19 MHz transmission
pulse in fundamental mode. Scale bars are 1 mm. Bottom panel: Fluorescence images of the agarose phantoms before and after
acoustic collapse; (d). Mean ultrasound and fluorescence signals from the GV samples tested in (c) (N ≥ 4, error bars are SEM);
(e). Diagram of a mouse implanted with a subcutaneous tumor model, and the expected spatial pattern of vascularization and
doxycycline-induced reporter gene expression; (f). Experimental timeline; (g). Representative ultrasound image of tumors con-
taining mARG-HEK cells after 4 days of doxycycline administration. mARG-specific contrast shown in the hot color map is
overlaid on an anatomical B-mode image showing the background anatomy; (h). Representative ultrasound image of tumors
containing mCherry-HEK cells after 4 days of doxycycline administration. The minimum and maximum values of color bars in
(g) are 4000 and 40,000 arbitrary units, respectively. Scale bars represent 1 mm for (g) to (h); (i). Representative power Dop-
pler images of a coronal cross section of the brain following GV injection. Scale bars, 2 mm; (j). Representative AM images of
a liver cross section following GV injection. Scale bars, 2 mm. Reproduced with permission from ref. 34, 40 and 51. (A higher
resolution/colour version of this figure is available in the electronic copy of the article).

resolution, giving this noninvasive and widely availa-
ble imaging technology a new function to visualize
bacteria that are genetically modified inside living an-
imals [39, 40].
4.2. GVs Gene Expression in Mammalian Cells
Furthermore, new attempts were carried out to make
GVs one of the molecular reporters that are genetically
encoded to connect ultrasound contrast with gene ex-
pression in mammalian cells. In 2019, a report de-
scribed how to engineer mammalian cells in order to
produce GVs [40]. For this purpose, a polycistronic
plasmid containing 8 P2A-tolerant GVs genes linked
by P2A sequences, a plasmid encoding GvpB, and a
polycistronic “booster” plasmid containing GvpJ,
GvpF, GvpG, GvpL, and GvpK were co-transfected
Biogenic Gas Vesicles for Ultrasound Imaging and Targeted Therapeutics
into HEK293T cells under a doxycycline-inducible
promoter to achieve stable expression of vesicles, sub-
sequent electron microscopy showed successful GVs
production. These three sets of gene structures are col-
lectively called mammalian acoustic reporter genes,
which can be sensitively detected even when the cell
volumetric density is less than 0.5%, enabling high-
resolution imaging of gene expression in live animals.
These HEK cells were then used to form model tumor
xenografts in immunocompromised mice. Four days
after induction, clear ultrasound contrast was observed
in the flank inoculated with mARG-HEK cells, which
area was consistent with subsequent histological exam-
ination of the tissue (Figs. 2e-h). This research is a step
forward in expanding the utility of GVs as imaging
tools, and more relevant work like condensing the con-
structs necessary for gas vesicle expression in mamma-
lian cells can be set out to facilitate such uses. There is
no wonder that further engineering of the genetic con-
structs comprising this acoustic reporter gene is ex-
pected to become a widely used reporting material like
green fluorescent protein.
4.3. GVs Gene Expression as Acoustic Biosensors
After making GVs expressed in mammalian cells,
researchers then focused on connecting the ultrasound
contrast of GVs to the activity of specific biomole-
cules. Based on GVs, Lakshmanan and his colleagues
developed the first genetically encodable acoustic bio-
sensors in response to the activity of protease enzymes
[48]. By engineering variants of GvpC that incorporate
amino acid sequences recognized by proteases, GvpC
can be shortened or totally removed by different prote-
ase enzymes, making GVs less rigid, thus buckling
more easily under acoustic pressure [34]. This reversi-
ble buckling can produce nonlinear contrast easily de-
tected by ultrasound. The innovative technique serves
as a bridge between ultrasound and activities of prote-
ase, and this research builds a future for visualizing a
variety of protease activities with noninvasive ultra-
sound imaging.
5. PRECLINICAL IMAGING APPLICATION OF
GVS
The US properties of GVs in vitro and in vivo have
been characterized by many researchers, and their safe-
ty has also been tested in different cells and animal
models, providing a glimpse of the potential of GVs to
serve as clinical ultrasound contrast agents. In 2017,
Johann Le Floc’h and his team investigated pharmaco-
kinetic properties and the clearance route of GVs by
radiolabeling them with 99mTc [49]. Single-photon
Current Medicinal Chemistry, XXXX, Vol. XX, No. XX 7
emission computed tomography showed that nearly 84
% of [99mTc]-GVs were taken up by the reticuloendo-
thelial system (RES) and 13 % were found in the gall
bladder and duodenum; the presence of GVs was rela-
tively for a short time in the blood. Therefore, re-
searchers made different modifications of GVs, and
improved the imaging technology, aiming to achieve
better targeted contrast enhancement. Here we intro-
duce several preclinical applications that have proven
to be feasible.
5.1. GVs for Transcranial Functional Ultrasound
Imaging
In 2019, Maresca et al. used GVs as a blood en-
hancer for functional ultrasound imaging of the mouse
brain, visually generating transcranial hemodynamic
contrast to depict changes in local cerebral blood vol-
ume caused by nerve activity at ultrafast frame rates
[50]. It has the advantages of high spatio-temporal res-
olution and versatile form factors. However, cranioto-
my is required to avoid the attenuation and distortion of
ultrasound caused by skull bones, which greatly limits
the scope of transcranial functional ultrasound imaging.
Injecting an ultrasound contrast agent into the cerebral
blood vessels can compensate for the attenuation of the
skull and solve this challenge to some extent. The re-
search team went further by comparing GVs with
commercial MBs in vitro and in vivo. The in vitro re-
sults showed that GVs could enhance Doppler contrast
in a wider speed range than microbubbles, which
means that these can detect slower flow more accurate-
ly, and can also withstand higher ultrasound pressure.
The in vivo results showed that GVs could provide a
quasi-steady-state hemodynamic contrast enhancement
similar to MBs, but the signal fluctuations are much
smaller. Further work can be set out to engineer GVs to
enhance circulation time or to generate stronger con-
trast, making GVs a more favored enhancer for tran-
scranial functional ultrasound imaging.
5.2. GVs for Ultrasound Molecular Imaging
Another application of GVs is ultrasound molecular
imaging for early tumor detection. In 2020, Sun Lei
and his colleagues reported a kind of targeted na-
noscale ultrasound molecular contrast agent by modify-
ing the surface of the GVs. Ultrasound molecular imag-
ing of living tumors requires suitable contrast agents.
The contrast agents need to have good biocompatibil-
ity, stability, small size, and targeting performance, and
they should not be easily cleared by the reticuloendo-
thelial system. Therefore, natural GVs need to be modi-
fied to meet the requirements mentioned above. Sun
8 Current Medicinal Chemistry, XXXX, Vol. XX, No. XX
Lei's team added hyaluronic acid (HA) and polyeth-
ylene glycol (PEG) on the surface of GVs in order to
obtain HA-GVs (hyaluronic acid modified) and PH-
GVs (Both HA and PEG modified). The PH-GVs are
not only more stable, but also have CD44-positive tu-
mor targeting capability via HA, which allows GVs to
selectively accumulate at the tumor site. Then SSC7
tumor-bearing mice were injected with GVs, HA-GVs,
and PH-GVs, respectively. The results showed that PH-
GVs showed much higher ultrasound intensity than the
other two GVs in tumor, and there was no cytotoxicity.
This improvement lays a solid foundation for the ultra-
sound molecular imaging of CD44 positive tumors by
taking advantage of with GVs [38].
5.3. GVs for Phagocytic and Lysosomal Activities
Recently, Shapiro and his team applied GVs in a
new field - to visualize and quantify phagocytic and
lysosomal activities in vivo by ultrasound imaging
(Figs. 2i, j) [51]. Traditional nanoscale ultrasound con-
trast agents usually did not undergo regular lysosomal
degradation. GVs, however, were mainly cleared from
circulation by liver-resident macrophages, typically by
Kupffer cells. Nonlinear ultrasound contrast showed
that the circulation half-life of GVs was 232 s, and ap-
parent half-life of GVs in the liver reached 20 min.
Then, after being internalized, the GVs were degraded
by lysosome, resulting in the elimination of their har-
monic ultrasound contrast. The phenomenon was then
used in the models of phagocyte deficiency and non-
alcoholic fatty liver disease, paving the way for nonin-
vasive functional imaging of cellular degradative pro-
cesses.
6. GVS FOR TARGETED THERAPEUTICS AND
RELATED MECHANISMS
6.1. GVs for Targeted Therapeutics
As described before, GVs are a unique class of ge-
netically encoded gas-filled protein nanostructures.
GVs have further been explored to own new abilities
such as reporter genes for a high-frequency ultrasound
and magnetic resonance imaging (MRI) [28, 39, 52].
Then, it was found that GVs can express in bacteria
[39] (Escherichia coli and Salmonella typhimurium)
and mammalian cells [39] by engineering the multi-
gene clusters encoding GVs. Like convention mi-
crobubbles, GVs show a series of biological effects,
which can be used not only for contrast imaging, but
also for drug/gene delivery or other therapeutic purpos-
es. In the following content, we would introduce some
of the latest advances of GVs as therapeutic agents,
which further expands the area of GVs applications.
Wang et al.
6.2. GVs as Nuclei for Cavitation
In a recent study carried out by Furusawa et al.,
GVs were found to serve as nuclei for the formation
and cavitation of free bubbles, turning targeted and
cell-expressed GVs into mechanical therapeutic war-
heads [53]. By using focused ultrasound of different
frequencies and an orthogonally positioned imaging
transducer, working as a passive cavitation detector,
researchers found that GVs purified from Ana can be
imaged safely when using typical diagnostic parame-
ters and are also able to serve as nuclei for inertial cavi-
tation at 0.67MHz. Ultra-high frame rate camera visu-
alized the process by which GVs nucleate the for-
mation of cavitating bubbles and gained consistent re-
sults, and further confirmed that GVs collapse at the
positive peak pressure, while the maximal growth of
the resulting bubbles occurs at the negative peak of the
ultrasound cycle. This kind of mechanical effect can
serve as the foundation of some future therapeutic ap-
plications. First, the all-protein shells of GVs make it
feasible to modify the surface of GVs. Chemical con-
jugation to GVs can be achieved by making use of ly-
sine residues on their protein shells and amine-reactive
cross-linkers such as sulfoN-hydroxysuccinimide esters
making it convenient for providing fluorescent GVs
and for GVs to be easily attached to particular cells.
Therefore, receptor-targeted GVs can serve as acousti-
cally detonated cellular disruptors, making an effort in
killing tumor cells. Second, genetic engineering of
GV’s mechanical and surface properties can be con-
veniently performed by exchanging the native outer
scaffolding protein GvpC (gas vesicle protein C) with
recombinant GvpC variants [20], achieving the hetero-
geneous expression of GVs in bacteria. Then the GV-
expressing bacteria could serve as efficient inertial cav-
itation nuclei, resulting in cell lysis and the release of a
co-expressed protein payload. According to the above
mechanism，Yan Fei and his colleagues made
achievement in providing a new tool for image-guided
and efficient gene delivery using GVs [54] . By cou-
pling with polyethylenimine (PEI), a cationic polymer
widely used for gene transfection, onto the shell of the
GVs, they synthesized a novel cationic biosynthetic
nanobubble as an ultrasonic gene delivery carrier,
which can effectively deliver genes into tumor cells
with the aid of ultrasound-targeted microbubble de-
struction. In their research, modified GVs significantly
improved gene transfection efficiency to 293T cells
and 4T1 cells when combined with ultrasound, provid-
ing a new gene carrier for image-guided gene therapy.
The above GVs applications bring together the targeted
therapeutics, genetic engineering technologies, focused
Biogenic Gas Vesicles for Ultrasound Imaging and Targeted Therapeutics
ultrasound, and inertial cavitation, providing a promis-
ing future. It is feasible to use ultrasound to visualize
the biodistribution of GVs, and then apply focused ul-
trasound to make inertial cavitation, achieving a thera-
peutic purpose.
6.3. GVs for Gas/Drug Carriers
Recently, GVs have also been applied as a new mo-
lecular oxygen carrier in oxygen-consuming cancer
therapy [55, 56]. The highly complex process of tumor
angiogenesis results in hypoxic conditions due to oxy-
gen transport within the tumor that is inadequate to
compensate for the high metabolic rate (Figs. 3a-d)
[57-59]. Compared to other synthetic nanobubbles, the
special characteristic of natural GVs excludes water but
allows gas to permeate in and out the protein shell
freely, thus creating a minimal pressure gradient, mak-
ing GVs significantly more stable. Moreover, as a ge-
netically encoded nanostructure, new epitopes or thera-
peutic ligands can be attached to the surface of GVs
through genetic recombinant approaches or chemical
conjunction approaches, gaining a better-targeted de-
livery to the tumor cells with high spatial and temporal
precision. Jean Gariépy and his team designed GVs as
therapeutic agents by attaching photoreactive chlorin
e6 (Ce6) molecules to lysine ε-amino groups on the
surface of purified Halobacterium sp. NRC-1 GVs,
designing light-activated GV nanoparticles called Ce6-
GVs (Figs. 3e, f). It is proved that this chemical conju-
gation of Ce6 to GVs improved the intracellular deliv-
ery into MCF-7 and FaDu cancer cells. When exposed
to light, Ce6-GVs were 200-fold more effective on a
molar basis than free Ce6 at killing cells, making them
a promising nanomaterial for image-guided photody-
namic therapy (Figs. 3g, h) [56].
Sun lei and his team also applied the GVs in tumor
therapy. They used lipid-coated GVs as a new stable
and effective oxygen carrier, overcoming the limita-
tions of coalescence, and short circulation time of other
synthetic bubbles. When combined with PDT (photo-
dynamic therapy), lipid-GVs significantly improved
ROS production and reduced viability of Human hepa-
toma cells, thus reducing tumor size in vivo and in-
creased the number of apoptotic cells. To sum up, en-
gineered GVs have the potential to be a kind of effec-
tive carrier, delivering oxygen or drug to the targeted
tumor sites when exposed to externally applied physi-
cal methods such as ultrasound or the light source,
leading a promising way to the era of precisely targeted
tumor therapies with better efficacies as well as little
side-effects.
Current Medicinal Chemistry, XXXX, Vol. XX, No. XX 9
6.4. Bioeffects Caused by Ultrasound
As described above, GVs have served as cavitation
nuclei, and there is no essential difference when com-
pared with conventional microbubbles in the mecha-
nisms. Ultrasound targeted microbubble destruction
technique can facilitate the transport of membrane im-
permeable compounds into cells and tissues via a series
of physical phenomena, such as inertial cavitation, liq-
uid jet, shear stress and microstreaming et al., contrib-
uting to translate applied ultrasound into local strain or
other energy forms [60]. Three kinds of bioeffects in-
fluence delivery efficiency and biosafety in this pro-
cess, sonoporation (plausible biological regulatory
mechanisms on membrane perforation and resealing),
interendothelial gap opening, and endocytosis en-
hancement post-cavitation [61]. Herein, we would give
a brief introduction to the three bioeffects.
6.4.1. Sonoporation
Sonoporation is termed as a transient and reversible
membrane perforation triggered by ultrasound-driven
microbubbles. By creating pores in cell membranes
using ultrasound, this mechanism can assist in facilitat-
ing the transport of molecules into or out of the cell
[62]. Both stable and inertial cavitation is involved in
this process [63, 64]. When the acoustic beam trans-
mits in a fluid that contains dissolved gases that can
form cavities, or stabilized microbubbles, the main ul-
trasound-induced mechanical stress arises [65]. These
gaseous inclusions respond to the acoustic cyclic pres-
sure alterations by oscillation to create micro-streaming
(defined as the formation of microflow around stably
oscillating microbubbles) and shear stresses in their
surrounding environment. This phenomenon is recog-
nized as ‘‘stable’’ cavitation. When high acoustic pres-
sures are used, the microbubbles experience violent
contraction and expansion, which ultimately results in
bubble collapse, releasing shear waves and liquid jets.
How much the cavitation of microbubbles affects near-
by cells and tissues relies much on the separating dis-
tance. The closer the targets to oscillating/imploding
microbubbles, the more violent the impact will be [66].
By focusing the acoustic beam at the objective loca-
tion, these mechanisms can improve the delivery of
molecules such as drugs and genes into target cells.
6.4.2. Interendothelial Junction Opening
Ultrasound-triggered delivery can momentarily alter
the vascular integrity and open the interendothelial
junction, thus forming an interendothelial gap, making
it easier for some macromolecular passing through the
endothelial barrier [67]. Previous studies observed the
10 Current Medicinal Chemistry, XXXX, Vol. XX, No. XX Wang et al.

Fig. (3). GVs for targeted therapeutics. (a). Representative photoacoustic images of tumor oxygen levels (Oxy-Hb and deoxy-
Hb levels) from in vivo tumor-bearing mice at different time points by tail vein injection of 200 µl of saline, PBS(O2), and 5
nM lipid-GVs(O2). Red pixels: oxy-Hb; blue pixels: deoxy-Hb; (b). Quantification of tumor oxygen levels. Data represent the
mean ± SD based on 4 independent experiments. ∗p < 0.05 vs. control; (c). Tumor growth curves of SCC7 tumor-bearing mice
with different treatment groups. n = 5 mice per group, ∗p < 0.05 significance level; (d). Body weight of SCC7 tumor-bearing
mice after various treatments; e,f. Transmission electron micrographs of (e) WT GVs and (f) Ce6-labeled GVs; (g, h). Dose-
dependent cell viability study of (g) MCF-7 cells or (h) FaDu-GFP cells treated with free Ce6 (closed triangles) or Ce6-GVs
(closed circles) and exposed to light as determined using the WST-1 cell viability assay. Results are shown as mean ± S.D. n =
3 from 2 independent experiments, with each concentration performed in triplicate. Reproduced with permission from ref. 55
and 56. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

spatiotemporal characteristics of the opening and clos-
ing of the interendothelial junction caused by ultra-
sound-driven microbubbles. When in the expanded
phase, the bubbles can cause a circumferential dis-
placement of the vessels, thus creating large gaps be-
tween cells, and that invagination of blood vessels oc-
curs in the contraction phase of the bubble, leading to
the delamination of the endothelial layer. It required
several minutes proximately to form the gaps between
endothelial cells. Once it was formed, the gap can be
remained for at least ten minutes under microbubbles
oscillation, providing a prolonged, enhanced vascular
permeability for effective gene and drug delivery [64].
The mechanism can be explained as follows. Firstly,
the interaction of shear stress from oscillating bubbles
with the centripetal and centrifugal forces from endo-
thelial cells may influence the cytoskeleton, focal adhe-
sion and interendothelial junctions, resulting in the en-
hancement of interendothelial permeability. Secondly,
when the microbubbles implode by ultrasound, it will
induce membrane perforation as well as the contraction
and shrinkage of surrounding cells, thus forming the
interendothelial gaps [68].
Biogenic Gas Vesicles for Ultrasound Imaging and Targeted Therapeutics Current Medicinal Chemistry, XXXX, Vol. XX, No. XX 11

6.4.3. Endocytosis Enhancement used imaging tool to in vivo cellular and biomolecular
function. Initial success leads the way of GVs to play a
Sonoporation and cavitation-opened interendothelial
more important role in ultrasound imaging and ultra-
gaps result in the formation of reversible pores on cell
sound guided/assisted therapy. Nevertheless, much
membranes, providing a passive diffusion way for
work should be done to ensure this nascent nanosized
small molecules. At the same time, cavitation-induced
bubble makes a brighter future in biology and medi-
endocytosis plays an important role in the active deliv-
cine. For example, a more intense study should be car-
ery route to assist the macromolecule to get into target-
ried out on the molecular biology and structural biolo-
ed cells. Several research works have found that two
gy of GVs, optimizing their acoustic properties to dis-
main routes were involved in the endocytosis progress:
tinguish their signal from background in ultrasound
clathrin-dependent endocytosis and caveolin-mediated
imaging maximally. Besides, researchers should focus
endocytosis [69]. Previous studies also reported that the
more on the molecular engineering of GVs to make
mechanism of particle internalization relied much on
them easily transferred to a variety of organisms, in-
the size of the molecule [70]. When the particle has a
cluding mammalian cells, thus monitoring and control-
diameter of < 200 nm, the molecules get into cells by
ling the process of cells as versatile acoustic reporter
clathrin-mediated endocytosis. When the size increased
genes when combined with ultrasound. Moreover, great
to 500 nm, caveolae-mediated internalization became
stress should be put on the concrete mechanisms of the
the predominant pathway [71]. Up to now, however, it
nucleated cavitation generated by GVs, making GVs
is not figured out yet what factors other than molecular
more prospective in targeted therapeutic applications.
size determine the delivery pathway into cells under the
Progress thus far has been exciting, and we expect a
influence of ultrasound-driven microbubbles. There-
brighter future of GVs in ultrasound imaging and tar-
fore, more research works are needed to clarify these
geted therapeutics.
problems further.
It is critical to deliver therapeutic agents across bio- LIST OF ABBREVIATIONS
logical membranes and endothelial barriers. However, GVs = Gas Vesicles
the complexity of living organisms makes it difficult to
GvpA = Gas Vesicle Protein A
achieve perfect specificity and avoid off-target effects.
Molecular targeting alone is often insufficient to direct C4F8 = Perfluorocyclobutane
systemically administered nanomedicines to desired GvpC = Gas Vesicle Protein C
anatomical locations. Fortunately, under the assistance
of cavitation nuclei such as microbubbles or GVs, ul- Ana = Anabaena Flos-aquae
trasound has the ability to open biological barriers, Halo = Halobacterium salinarum NRC-1
which have been used for site-specific delivery of mol- AM = Amplitude Modulation
ecules, nanoparticles, and viral vectors to tissues such
as the brain [72], the eye, muscle, the gastrointestinal PI = Pulse Inversion
tract, and tumor [73, 74]. Several reviews have covered CNR = Contrast-to-Noise Ratio
the application and bioeffects of ultrasound-driven mi-
CAR = Contrast-to-Artifact Ratio
crobubbles to enhance the macromolecule delivery,
aiming to leverage a deeper understanding of these bio- xAM = Cross-Amplitude Modulation
logical mechanisms [75-78]. CTR = Contrast-to-Tissue Ratio
CONCLUSION ST = SpyTag
As it is described in this review, ultrasound has been SC-MNG = SpyCatcher-mNeonGreen
developed quickly, providing brand new ideas for the
GFP = Green Fluorescent Protein
diagnosis and treatment of diseases, observing and
monitoring the biological functions of living organics ECN = E. coli Strain Nissle 1917
due to its unique advantages of physic functions and RES = Reticuloendothelial System
tissue interactions. Biomolecular agents serve as the
bridge in connecting ultrasound with specific biomole- HA = Hyaluronic Acid
cules, cells and tissues, thus making it possible to im- PEG = Polyethylene Glycol
age and control cellular processes precisely. In this
MRI = Magnetic Resonance Imaging
field, GVs show great promise to connect this widely
12 Current Medicinal Chemistry, XXXX, Vol. XX, No. XX
PEI = Polyethylenimine
Ce6 = Chlorin e6
PDT = Photodynamic Therapy
CONSENT FOR PUBLICATION
Not applicable.
FUNDING
The authors gratefully acknowledge the support of
the National Natural Science Foundation of China
[grant numbers 82171964, 81530056, 81922033,
81671705], and the Graduates’ Innovation Fund,
Huazhong University of Science and Technology
(2021yjsCXCY125).
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial
or otherwise.
ACKNOWLEDGEMENTS
Rui Wang and Lufang Wang contributed equally to
this work.
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