Biomaterials 133 (2017) 154e164

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Review

Recent advances in CO2 bubble-generating carrier systems for

localized controlled release
Yu-Jung Lin a, 1, Chieh-Cheng Huang b, 1, Wei-Lin Wan a, Ching-Hua Chiang a, Yen Chang c,
Hsing-Wen Sung a, b, *
a
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC
b
Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC
c
Department of Cardiovascular Surgery, Heart Center, Cheng Hsin General Hospital and College of Medicine, National Yang-Ming University, Taipei, Taiwan,
ROC

a r t i c l e i n f o a b s t r a c t

Article history: This article reviews recent progress in the development of carbon dioxide (CO2) bubble-generating drug
Received 19 January 2017 carriers, including their designs and operating mechanisms; these carriers constitute an advanced class
Received in revised form of stimuli-responsive delivery systems with considerable potential. The drug carriers contain stimuli-
8 April 2017
responsive agents, which are stable before they reach the target location, but enable rapid drug
Accepted 12 April 2017
Available online 12 April 2017
release that is triggered by the generation of CO2 bubbles, which are chemically inert, under certain
stimuli. These CO2 bubble-generating carrier systems can be used to accumulate locally a delivered drug
at the diseased tissue, while reducing side effects on the normal tissue, improving their therapeutic
Keywords:
Stimuli-responsiveness
effectiveness. Since the generated CO2 bubbles are hyperechogenic, they may also be used as an ultra-
Gas bubble sound contrast agent in elucidating the status of the carriers and providing real-time diagnostic images.
Ammonium bicarbonate Perspectives of the future of applications of gases with therapeutic effects, such as nitric oxide (NO),
Sodium bicarbonate carbon monoxide (CO), and hydrogen sulde (H2S), in such bubble-generating carrier systems, are also
Calcium carbonate briey discussed.
2017 Elsevier Ltd. All rights reserved.

1. Introduction To increase its therapeutic efcacy, a stable carrier system must

Delivering a therapeutic cargo using a carrier system specically
to the site of a disease and then releasing a drug locally on demand
without affecting the normal tissue is a formidable undertaking
[1e4]. An efcient carrier system should lose a negligible amount of
the drug in its storage and delivery stages, and rapid drug release at
a concentration within its therapeutic window is required after the
carrier is delivered to the target tissue [1,5,6]. To prevent the pre-
mature release of the therapeutic drug before delivery to the
location of the disease, the carrier system must be stable [1,6,7].
However, in the absence of a triggering mechanism, the amount of
drug that is released from a stable carrier system is usually limited
[1,8e10]. Therefore, the concentration of the drug that accumulates
in the diseased tissue may fail to reach the therapeutic threshold.
* Corresponding author. Department of Chemical Engineering, National Tsing
Hua University, Hsinchu 30013, Taiwan, ROC.
E-mail address: hwsung@mx.nthu.edu.tw (H.-W. Sung).
1
The rst two authors (Y.J. Lin and C.C. Huang) contributed equally to this work.
also exhibit stimuli-responsive characteristics that enable the
active triggering of drug release such that its local concentration
can be controlled within the therapeutic window [11]. A range of
stimuli such as endogenous pH and biomolecules, and exogenously
applied temperature, light, and ultrasound have been used to
destabilize the carrier system, creating a stimuli-responsive drug
release [1,2,12e16].
Many polymeric materials have been used to fabricate stable
vehicles for carrying therapeutic drugs [11,17,18]. Some of these
polymeric materials are themselves stimuli-responsive, meaning
that they can change their conformation or physicochemical
properties upon encountering specic environmental cues, trig-
gering drug release [19]. However, most conventional materials
that have been proposed in the literature are insufciently sensitive
to respond rapidly to small changes in physical or chemical con-
ditions in the local environment to ensure appropriate spatial,
temporal, and dosage control. A novel method has recently been
developed to realize spatially, temporally, and dosage-controlled
release; the method uses stimuli-responsive agents, in the

http://dx.doi.org/10.1016/j.biomaterials.2017.04.018
0142-9612/ 2017 Elsevier Ltd. All rights reserved.
 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164
vehicles, that can generate carbon dioxide (CO2) bubbles upon
exposure to an exogenous temperature, an endogenous pH, or
reactive oxygen species (ROS) [8e10,20e35]. The method provides
a means of destabilizing the carrier for localized controlled release.
CO2 is generally regarded as a non-toxic, chemically inert sub-
stance, which is highly soluble in blood and tissue, and so that its
gas emboli can be readily absorbed [36]. CO2 has been extensively
used clinically as an insufation gas in laparoscopic procedures for
the diagnosis and treatment of intra-abdominal and gynecological
diseases [36e38].
This review focuses on the recent development of CO2 bubble-
generating carrier systems for localized controlled release, with
emphasis on their designs, operating mechanisms, and applica-
tions. Table 1 presents experimental details (including carrier ma-
terials, CO2 generators, triggering methods/mechanisms, payloads,
applications, cell types, and animal models) in each example. The
limitations of these CO2 gas-generating systems and those that
potentially generate other gases with therapeutic effects are also
discussed.
2. Thermoresponsive drug release
Ammonium bicarbonate (ABC, NH4HCO3) is used as a raising
agent to produce gas bubbles in the baking of goods [39]. Upon
heating, ABC decomposes to produce CO2 bubbles; the process
begins at a temperature of approximately 36
C and complete
decomposition is achieved at about 60
C [40e42]. In the human
body, bicarbonate ions (HCO 3 ) are used to balance physiological
pH, before being released by the lungs as CO2 [43,44]. When
encapsulated in a carrier system, ABC may serve as a thermores-
ponsive agent for triggering localized drug release upon the
application of heat using a hot-water mat [23], near-infrared (NIR)
light [25], or ultrasound [45].
2.1. Applications in extracellular release
Liposomes (Lips) have been widely exploited as an extracellular
or intracellular delivery system for treating cancer because their
surfaces are easily modied [20,46]. The decomposition of ABC to
generate CO2 bubbles was rst exploited to trigger extracellular
drug release from Lips that contained doxorubicin (DOX) in a locally
heated tumor [22,23]. This bubble-generating method also pro-
vided a unique means forming in situ a oating hydrogel that
controllably released a drug to treat bladder cancer [29].
2.1.1. Generation of CO2 to trigger localized drug release
PEGylated Lips have been applied as stable carriers for trans-
porting DOX, known as Doxil, which has been used as a chemo-
therapeutic medication for a variety of tumors [47,48].
Nevertheless, the slow and passive release of a drug from PEGylated
Lips (<10% in 24 h) greatly limits its chemotherapeutic ability
[49,50]. Therefore, a means of actively activating the release of a
drug from Doxil is required.
To achieve the aforementioned goal, a thermoresponsive
bubble-generating Lip formulation was recently reported [22,23].
The key component of this Lip formulation was its encapsulated
ABC (ABC Lips), which performs two major functions (Fig. 1). Firstly,
the encapsulated ABC can be used with a remote loading technique
to establish the transmembrane gradient that is required for the
highly effective encapsulation of DOX in the aqueous compartment
of the Lips [22]. Secondly, the encapsulated ABC can be utilized to
activate swift drug release from the Lips in response to mild
heating. To prevent damage to the cells, the hyperthermic tem-
perature applied in the study was limited to 42
C. Reportedly,
cells typically undergo apoptosis or mitotic death when tissues are
155
heated above 45
C [51].
Upon heat treatment, ABC rapidly decomposed to form CO2
bubbles, generating leaky defects in the lipid bilayer of the Lips,
quickly releasing DOX, and thereby producing a high extracellular
drug concentration (Fig. 1). Following diffusion into a cell and ul-
timately moving into its nucleus, DOX molecules bond effectively to
nuclear DNA, triggering the death of the cell. Mild heat treatment
can improve tumor perfusion and enhance the sensitivity of the cell
to the drug [52].
2.1.2. In situ formation of a oating hydrogel by CO2 generation
ABC decomposition to form CO2 bubbles at high temperature
was also used in preparing a hydrogel system that could oat in the
bladder as an in situ drug delivery system without causing a urinary
obstruction [29]. This drug delivery system consisted of an instil-
lable mixture of a thermoresponsive polymer (Poloxamer 407), an
anticancer drug (DOX), and a bubble-generating agent (ABC), which
was in a solution form at room temperature. Following instillation
into a rabbit bladder, the polymeric solution transformed to the gel
state at body temperature, oating in the bladder owing to the
production of CO2 bubbles, enabling the localized release of DOX
(Fig. 2). Empirical results revealed that this oating hydrogel can be
used as an intravesical delivery system to reduce the rate of
recurrence of the bladder tumor following transurethral resection
[29].
2.2. Applications in intracellular release
The Lips that can generate CO2 bubbles under mild heating were
also used to induce cell necrosis after they had been internalized
and undergone endocytosis and intracellular trafcking to lyso-
somes [20]. As part of a ligand targeting strategy, this bubble-
generating Lip platform was used in the intracellular delivery of
an anticancer drug to a particular cellular organelle [25]. A similar
bubble-generating Lip system that can be thermally triggered to
liberate instantly and precisely its loaded iron oxide nanoparticles
(IONPs) in specied cellular organelles was utilized to elucidate the
mechanism of the cellular organelle-dependent cytotoxicity of the
IONPs [26].
2.2.1. Generation of CO2 for inducing cell necrosis
In conventional chemotherapy, after an antitumor agent has
killed tumor cells, the agents that remain may harm normal cells
and tissues. To solve this problem, a thermally responsive Lip sys-
tem that contained the bubble-generating agent ABC without any
antitumor drug that could kill tumor cells by generating CO2 bub-
bles rapidly was developed [20]. Fig. 3 schematically depicts this Lip
system and its function. The Lips can be readily internalized by the
cancer cells in an electrostatic interaction between their positive
surface charge and the intrinsic negative charge of the cell mem-
brane. Following endocytosis and intracellular trafcking to lyso-
somes, the Lips that contained ABC were thermally activated at
42
C to form CO2 bubbles, which grew quickly and collapsed
ercely to produce a disrupting force that is similar to the cavitation
effect stimulated by ultrasound [53], disturbing the lysosomal
membrane and releasing lysosomal proteases, causing cell necrosis
without leaving behind any cytotoxic agents. Accordingly, only the
cells that internalized Lips were destroyed when heated, while
their neighboring cells remained unharmed.
Zhang et al. reported on the concept of using a CO2 bubbling-
based system as a nanobomb to cause the cavitation-induced
necrosis of tumor cells using hollow mesoporous silica NPs that
contained L-arginine (LA) that could reversibly adsorb and release
CO2 gas in response to the environmental temperature [45]. Low-
intensity ultrasound was applied in vitro and in vivo to produce
 156
Table 1
A summary of experimental details in each study that is reviewed in this article.

Carrier CO2 Triggering Methods/ Payloads Applications Experimental Models References

Materials Generators Mechanisms
Cell Types Animal Models

Thermo-responsive Applications in Liposome ABC Water mat DOX Tumor inhibition H460 cells BALB/c nude mouse 22, 23
Drug Release Extracellular Hydrogel ABC Body temperature DOX Tumor inhibition N/A Rabbit 29
Release

Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164

Applications in Liposome ABC Water mat DOX Tumor inhibition MCF-7/ADR cells BALB/c nude mouse 9
Intracellular Liposome ABC Water bath N/A Tumor inhibition HT1080 cells N/A 20
Release Liposome ABC NIR DOX, Au NGs Tumor inhibition MCF-7 cells BALB/c nude mouse 25
Liposome ABC Water bath IONPs Tumor inhibition HT1080 cells N/A 26
Liposome ABC NIR DOX Tumor inhibition KB cells, A549 cells BALB/c nude mouse 27
Liposome ABC NIR DOX Tumor inhibition MDA-MB-231 cells NCr nude mouse 28
Mesoporous L-arginine Low-intensity N/A Tumor inhibition Panc-1 cells New Zealand rabbit, 45
silica ultrasound nude mouse
pH-responsive Drug Environmental pH- Hollow PLGA SBC Low pH at lysosomes DOX Tumor inhibition Hep3B cells N/A 10
Release triggered Generation of Hollow PLGA SBC Low pH at local Vancomycin Suppression of None New Zealand rabbit 24
CO2 inammatory site inammation
Hollow SBC Low pH at lysosomes DOX Tumor inhibition MCF-7 cells, MCF-7/ N/A 30
mesoporous ADR cells
silica
Hollow PLGA ABC Low pH at endosome Ovalbumin Immune response Dendritic cells, T cells C57BL/6 mouse 33
and lysosome enhancement
CaCO3 CaCO3 Low pH at tumor site DOX Tumor inhibition, SCC-7 cells C3H/HeN nude mouse 76
ultrasound
contrast enhancement
Exogenously Controlled Hollow PLGA SBC High ROS levels at DEX-P Suppression of N/A BALB/c mouse 8
pH-triggered inammatory site inammation
Generation of CO2 CaCO3 CaCO3 Protonated tranexamic Thrombin Halting hemorrhage N/A BALB/c mouse, porcine 82
acid

ABC: ammonium bicarbonate; SBC: sodium bicarbonate; NIR: near-infrared; DOX: doxorubicin; ROS: reactive oxygen species; Au NGs: gold nanocages; DEX-P: dexamethasone sodium phosphate; N/A: not available.
 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164 157

Fig. 1. Structure and functions of thermoresponsive, bubble-generating liposomes (Lips) and mechanism of localized extracellular drug release that is triggered by heat. ABC:
ammonium bicarbonate; DOX: doxorubicin. Reprinted with permission from Ref. [22].

hyperpyrexia locally, generating a large amount of CO2 bubbles; 2.2.2. Generation of CO2 for triggering drug release at lysosomes
meanwhile, the shock wave of the applied ultrasound caused an Stimuli-responsive Lips that can be remotely triggered to release
instantaneous implosion of the generated CO2 bubbles, inducing their cargo at a particular cellular organelle in cancer cells, such as
cavitation-mediated tumor destruction [45]. lysosomes, have received much attention in chemotherapy,

Fig. 2. Schematic illustrations of a CO2 bubble-generating hydrogel system that can oat in the bladder as an in situ drug delivery system without causing a urinary obstruction.
158 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164

Fig. 3. Structure of Lips that generate bubbles upon heating, and their use in rupturing cancer cells by transient cavitation, resulting in cell death. Reprinted with permission from
Ref. [20].

particularly for the purpose of overcoming multidrug resistance time for the NIR-induced heat-activated release of the drug after
(MDR). In spite of the superior anticancer activity of DOX, its ef- systemic administration of the Lips (Fig. 4). The obtained experi-
ciency is severely restricted by the MDR of tumor cells [54]. To mental results demonstrated that the targeted delivery of both heat
overcome this cellular MDR effect, several Lip formulations that and the drug to the cancer cells by the reported multifunctional Lip
contained ABC and DOX, with a targeted ligand that was conjugated system had the potential to maximize antitumor activity [25].
on their surfaces, were presented [9,27]. Functionalization with a
ligand can improve the afnity of the Lips for, and their specic
2.2.3. Mechanistic study of cellular organelle-dependent
binding to, the receptors on tumor cells, favoring their following
cytotoxicity of IONPs
internalization. After endocytosis and intracellular trafcking to
IONPs have commonly been used to label cells for cellular sep-
lysosomes, a mild hyperthermia at 42
C was externally applied. In
aration, sorting [55,56], and tracking [57,58]; they have also been
response to the hyperthermia, the decomposition of ABC that was
widely used in the diagnosis and treatment of tumors [59e62].
encapsulated in the Lips enabled the instant production of CO2
However, conicting results with respect to the cytotoxicity of
bubbles, destabilizing the Lip membrane, enabling the rapid release
IONPs have been reported [63e66]. The most commonly offered
of DOX in cells, and thereby overcoming the MDR effect. The swift
hypothesis concerning IONP toxicity involves the pH of the envi-
intracellular release of the drug considerably increased the amount
ronment to which IONPs are exposed during endocytotic trans-
of DOX that subsequently accumulated in the nuclei above the
portation [59,63,67]. To test this hypothesis, Lip vehicles that
threshold for triggering cell death by apoptosis [9]. Furthermore,
contained IONPs and ABC that could generate CO2 bubbles and
the membranes of the lysosomes were disturbed, causing them to
would not cause signicant cell death were used [26]. Following
release proteases into the cytoplasm, resulting in the necrosis of
cellular uptake by endocytosis, the internalized Lips were thermally
cells [20].
triggered at 42
C in various stages of the process of intracellular
Although the aforementioned thermoresponsive Lip platform is
transportation to liberate instantaneously their loaded IONPs in
promising for the treatment of cancer, the optimal time for the
particular acidic organelles (Fig. 5).
in vivo heat-activation of drug release after the systemic delivery of
Intracellular pH values in cytosol, endosomes, and lysosomes
Lips is unknown. To address this issue, a multifunctional Lip system
are known to be 7.2, 6.0, and 5.0, respectively [68,69]. In acidic
with an activatable molecular beacon (a hybridized Mucin-1
environments, IONPs, which mostly comprise Fe3O4 [59], can be
aptamer) that can deliver an anticancer drug (DOX), an NIR-
degraded by hydrolysis into free iron ions (Fe2/Fe3) [70]. Higher
inducible heat source (gold nanocages), and a bubbling agent
IONP toxicity was associated with stronger in situ degradation with
(ABC) into targeted cancer cells to exert a cytotoxic effect was re-
the release of more iron ions, and the consequent formation of
ported [25]. The activatable molecular beacon can be used to
more ROS within cells. When the amount of formed ROS exceeded
monitor the in vivo dynamics of the accumulation of the Lips in the
the amount that could be scavenged by the intracellular antioxi-
tumor in real time, facilitating the determination of the optimal
dant systems, the cells experienced oxidative stress, which was
 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164 159

Fig. 4. Composition and structure of an h-MUC1 AuNG-Lip, and mechanism of its simultaneous delivery of heat and DOX into a tumor cell for spatially and temporally precise
targeted drug delivery. AuNG: gold nanocage; h-MUC1: hybridized Mucin-1 aptamer. Reprinted with permission from Ref. [25].

Fig. 5. Structure of thermoresponsive bubble-generating liposomal system and process of its spatially precise, controlled intracellular liberation of IONPs in particular cellular
organelles in various endocytotic stages. The degradation of IONPs, release of iron ions, and subsequent reactive oxygen species (ROS) generation within cells are indicated. IONPs:
iron oxide nanoparticles; DMT1: divalent metal transporter-1. Reprinted with permission from Ref. [26].

responsible for the observed cellular organelle-dependent toxicity
proles. Understanding the mechanism that underlies the toxicity
of IONPs is crucial in designing IONP nanosystems with a wide
range of clinical applications [26].
3. pH-responsive drug release
Sodium bicarbonate (SBC, NaHCO3) is the most commonly used
buffer in standard cell culture media. Mammalian tissues are
immersed in an environment that normally contains bicarbonate
160 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164
Fig. 6. Structure of a PLGA hollow microsphere that contains doxorubicin (DOX), mechanism of its drug release, and intracellular trafcking and release of drug from pH-responsive
microspheres. Reprinted with permission from Ref. [10].
ions (HCO3 ), and cells have developed a mechanism to internalize
extracellular HCO 3 to neutralize their cytoplasm [69]. In acidic
environments, SBC decomposes spontaneously, rapidly forming
CO2 bubbles [71]. Therefore, when incorporated into carriers, SBC
can be used as a stimulating agent to trigger localized drug release
by generating CO2 bubbles as determined by their pH
microenvironments.
3.1. Environmental pH-triggered generation of CO2
The pH value of the typical extracellular milieu is 7.4, and
intracellular organelles are characterized by the gradual acidica-
tion of their compartments as the they mature from early endo-
somes into late endosomes and lysosomes [68,69]. Furthermore,
some diseased tissues, including tumors, or areas of infection,
inammation, and ischemia exhibit abnormally acidic extracellular
environments [72,73].
3.1.1. Drug release triggered in acidic cellular organelles
Poly(D,L-lactic-co-glycolic acid) (PLGA) has been extensively
utilized for delivering drugs because of its superior biocompati-
bility and biodegradability. However, the degradation of PLGA-
based carriers is typically slow, limiting their therapeutic efcacy
[74]. This shortcoming motivated the development of a hollow
microsphere (HM) system that can deliver an anticancer drug into
cancer cells and swiftly release the drug within acidic organelles
such as lysosomes [10]. The HMs were constructed from PLGA
employing a double-emulsion method, with the aqueous core that
contained an anticancer drug DOX and an acid-stimuli gas-gener-
ating agent SBC.
Once tumor cells had internalized HMs and transported them to
the lysosomal compartments, the protons (H) that inltrated the
HMs from the acidic organelles reacted with the SBC therein,
rapidly forming CO2 bubbles (Fig. 6). The evolution of CO2 bubbles
triggered the bursting of the PLGA shell wall by increasing the in-
ternal pressure, swiftly releasing DOX locally and promoting the
following accumulation of DOX in the nuclei. Such a pH-responsive
bubble-generating system, which could bypass the drug efux that
is mediated by P-glycoprotein transporters, was also utilized in the
controlled and rapid release of drug in acidic organelles to over-
come the MDR effect, increasing the therapeutic efcacy of the drug
against cancer [30,31,35].
To overcome the MDR of tumor cells, pH-responsive bubble-
generating NPs that contained no anticancer drugs but could
spontaneously generate CO2 bubbles in the acidic environment of
lysosomes were developed [30]. After they had been internalized
by tumor cells and transported to lysosomes, the rapidly generated
CO2 bubbles effectively increased lysosomal membrane per-
meabilization. Consequently, certain cathepsins were released from
the lysosome into the cytoplasm, enhancing cell death and
considerably increasing the efciency with which MDR was
mitigated.
The evolution of CO2 bubbles in response to the environmental
pH, triggering the release of the payload, was exploited to develop
an NP-in-microsphere hybrid delivery system for the co-delivery of
minicircle DNA and an anticancer drug to kill cancer cells [32].
Similar concepts were reported in the design of PLGA-based hollow
NPs that can quickly release antigens in dendritic cells, which are
the most potent and critical antigen-presenting cells, to improve
the quality and magnitude of a vaccine response [33].
3.1.2. Drug release triggered in acidic tissue environment
SBC was used as a pH-responsive bubble-generating agent to
trigger local drug release in the acidic environment that is pro-
duced by inammation in cases of osteomyelitis [24]. In osteomy-
elitis, the production of a great amount of acid by infecting bacteria
reduces the local pH down to approximately 5.4 [75], which can
stimulate the local unloading of an antibiotic from a carrier system.
To ensure that the local antibiotic concentration sufced for
treating osteomyelitis, an injectable calcium phosphate cement
that contained pH-responsive PLGA HMs that could control the
release of a drug in a manner determined by the local pH was
developed [24]. The HMs were constructed using a microuidic
device to have a shell of PLGA and an aqueous core that contained
 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164 161

Fig. 7. Composition and structure of injectable CP/HM composite cement that is developed herein and mechanism of its involvement in treatment of osteomyelitis. CP: calcium
phosphate; HM: hollow microsphere; Van: vancomycin. Reprinted with permission from Ref. [24].

an antibiotic vancomycin and SBC. After they had been mixed with
water, the calcium phosphate and HMs became a paste that was
injectable and conformed to bone defects in osteomyelitis (Fig. 7).
In acidic, inamed tissues, the SBC that was encapsulated in the
HMs quickly reacted with the local acid to form CO2 bubbles,
disturbing the HM wall and thus promptly releasing an amount of
encapsulated vancomycin that exceeded the therapeutic threshold
for curing the disease.
Not only is an acidic environment known to be endogenously
present in inamed tissues, but also the extracellular pH of tumors

Fig. 8. Composition and structure of ultrasensitive ROS-responsive gas-generating HM and mechanism of its involvement in treatment of osteoarthritis. ROS: reactive oxygen
species; DEX-P: dexamethasone sodium phosphate; SBC: sodium bicarbonate. Reprinted with permission from Ref. [8].
162 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164
(6.8e7.2) is known to be lower than that of normal tissues (pH 7.4)
[76], and this pH can be chosen as a target for treatment. In an
acidic milieu, inorganic mineral calcium carbonate (CaCO3) can
react with protons and dissolve to form CO2 gas [77]. A CaCO3
mineralized NP system that can generate CO2 bubbles and trigger
the release of its encapsulated DOX at the tumoral acidic pH was
proposed, exploiting this unique feature [76]. Thus generated CO2
bubbles may also serve as an effective echogenic contrast agent,
providing a theranostic platform for cancer therapy.
3.2. Exogenously controlled pH-triggered generation of CO2
As discussed above, the decomposition of SBC under the pH
conditions in intracellular lysosomes (pH 5.0) and extracellularly in
cases of osteomyelitis (pH 5.4) both generated enough CO2 bubbles
to trigger effectively the release of a drug with a concentration
within its therapeutic window. However, in cases of osteoarthritis,
the pH in inamed joints reaches only approximately 6.6e7.1 [78],
which may not be sufciently low to create an adequate CO2
pressure to trigger the release of enough of a drug to cure the
disease. During osteoarthritic inammation, the local production of
ROS is promoted, with a biologically relevant concentration of H2O2
of around 50e100 mM [79,80]. H2O2 is the precursor for the pro-
duction of most ROS [81], which have been suggested to be stim-
ulus targets in the design of triggered drug release systems [8,34].
An ultrasensitive ROS-responsive HM carrier that contained an
anti-inammatory drug (dexamethasone sodium phosphate, DEX-
P), an acid precursor that comprised ethanol and FeCl2, and SBC,
was developed to treat osteoarthritis locally, by taking advantage
its enhancement of the local production of H2O2 even at a very low
concentration [8]. Upon injection into the inamed joint, a low
concentration of H2O2 diffused into the HMs, oxidizing their
encapsulated ethanol in the presence of Fe2, via the Fenton reac-
tion, to form acetic acid, further reducing the local pH exogenously
(Fig. 8). Therefore, the decomposition of SBC in the enhanced acidic
environment was signicantly improved so many CO2 bubbles
were formed, triggering the release of DEX-P from the HMs,
providing a high dose of the anti-arthritic drug to protect against
arthritis and joint destruction.
Delivering a therapeutic coagulant deep into damaged tissue to
clot the outwardly owing blood during bleeding is difcult. To
overcome this challenge, gas-generating CaCO3-based microparti-
cles that carried a hemostatic agent, thrombin, and could actively
transport against blood through wounds and into the vasculature
were developed [82]. CaCO3 is frequently used in antacid tablets
[83], as it is stable and insoluble in the neutral aqueous environ-
ment, but rapidly decomposes to form CO2 bubbles under acidic
conditions [84]. When CaCO3 microparticles were mixed with
protonated tranexamic acid, they rapidly traveled through blood,
being propelled by the spontaneous generation of CO2 bubbles in
the acidic pH environment that was exogenously controlled by the
organic acid, halting severe hemorrhaging in multiple animal
models [82]. The ndings of the present investigation revealed that
the self-propelling particles can function as an active delivery
system that transports agents upstream through blood.
4. ROS-responsive drug release
The induction and development of many diseases such as the
ischemia/reperfusion injury, inammation, hypertension, and
cancer generally involve the overproduction of ROS, including H2O2
[85]. Utilizing ROS as a diagnostic biomarker and therapeutic target
is therefore a promising approach to the diagnosis and treatment of
such diseases. An H2O2-responsive poly(vanillin oxalate)-based NP
system that could react with H2O2 to form echogenic CO2 bubbles
and the antioxidant vanillin was recently reported to be effective in
treating hepatic ischemia/reperfusion injury in a mouse model
[34]. The relevant experimental results suggested that this unique
method has great potential to provide diagnostic ultrasound im-
ages of ROS-associated diseases and improve their therapeutic
efcacy.
5. CO2 bubbles as an ultrasound contrast agent
Ultrasound is a non-invasive, safe, and convenient tool that can
provide biomedical images in real time; to improve the intensity of
the images that it yields for the purpose of clinical diagnosis,
several contrast agents in the form of microbubbles have been
developed [86,87]. Owing to their high echogenic sensitivity, CO2
bubbles have recently attracted much attention as an efcient ul-
trasound contrast agent. As an example, CO2 bubbles have been
used clinically in contrast-enhanced sonography to differentiate
inammatory masses from ductal carcinomas of the pancreas [88].
Polycarbonate particles that can generate CO2 bubbles by the
hydrolysis of their carbonate side chains have been used to amplify
the echo reectivity of tumor tissue in mice [86], the subcutaneous
area of the back in mice, and the heart in rats [87]. Furthermore,
CO2 bubbles that are generated by CaCO3 NPs have been shown to
provide high and long-lasting echogenicity in tumor tissues with
low pH [77]. In acidic solutions, the NPs of CaCO3 decompose
continuously to form CO2 bubbles, making them an effective ul-
trasound contrast agent.
6. Limitations of CO2-generating carrier systems
Intravascular CO2 embolism can be problematic, although it
occurs rarely in laparoscopic procedures. Intravascular entrapment
of CO2, resulting in blockage of the right ventricle or pulmonary
artery, has been reported [38]. When these CO2-generating carrier
systems are triggered in a vein or artery, gas embolism may occur,
occluding blood vessels. Additionally, the accumulation of such
bubble-generating systems in the lungs may be detrimental as they
can disrupt gas exchange. To prevent potential embolization,
extravascular triggering of these CO2-generating carrier systems is
favored.
7. Conclusions and future perspectives
Considerable efforts have recently been devoted to the design
and assembly of many CO2 bubble-generating carriers and to study
their potential uses in stimuli-responsive delivery systems for
treating various diseases and as ultrasound contrast agents for
providing real-time images for clinical diagnosis. These bubble-
generating carriers may serve as advanced stimuli-responsive de-
livery platforms for the control of localized drug release from stable
carriers following their accumulation at the site of diseases, in
response to changes in their surrounding environment.
Since CO2 is chemically unreactive, it has no therapeutic effects.
Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulde
(H2S) are gasotransmitters, which are crucial to the modulation of
various physiological functions in the mammalian body [89]. NO is
a cell-signaling molecule that can regulate many biological events,
including vasodilation, angiogenesis, and smooth muscle contrac-
tion [90]. CO is a messenger that has anti-inammatory and anti-
apoptosis effects [91], and H2S participates in immune reactions,
cardiovascular protection, and antioxidation [92]. As well as criti-
cally triggering drug release, like biologically inert CO2, these
gasotransmitters may act as therapeutic agents in the modulation
of a multitude of physiological functions. Given the exciting ad-
vances that are mentioned above, we believe that these bubble-
 Y.-J. Lin et al. / Biomaterials 133 (2017) 154e164
generating carriers may lead to signicant technological break-
throughs in the treatment and diagnosis of various human pa-
thologies in the near future.
Acknowledgements
This work was supported by grants from the Ministry of Science
and Technology (MOST 105-2119-M-007-008) and National Health
Research Institutes (NHRI-EX106-10522EI), Taiwan (ROC).
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