Ultrasound-responsive microbubbles in antibacterial therapy

Abstract

Microbubbles (MBs) are gas-filled micrometer-scale spheres that are commonly formed by the gas core encapsulated with stabilizing shells, including polymers, surfactants, proteins, or liposomes shells. Clinically, MBs were originally used as contrast agents for enhanced ultrasound (US) imaging and diagnostics. Nowadays, MBs were given expectations that they can be alternative platforms for drug delivery owing to their unique acoustic properties. MBs can respond to the US by cavitation effect which refers to a series of complex dynamic processes, such as oscillation, expansion, contraction, and implosion [1]. Drug molecules or therapeutic agents can be associated with the MB shells by means of van-der-Waals forces, electrostatic or hydrophobic interactions, or merely by physical encapsulation [2]. Therefore, strategies are emerging which take advantages of US-mediated MBs drug delivery systems, mainly focusing on sonothrombolysis, cancer therapy and central nervous system (CNS) pathologies [3]. Nevertheless, several researchers have apperceived the promising potential of US-responsive MBs in antibacterial therapy. Here, we aimed to paint an overview of the latest published papers on MBs for antibacterial therapy, hoping to help understand the perspectives that the field may offer emerging generations of antibacterial agents.

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Ultrasound-responsive microbubbles in antibacterial therapy
Xiao-Ye Li1, Wei-Jun Xiu2, Dong-Liang Yang3, Heng Dong1*
1Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China. 2Key Laboratory for Organic Electronics and
Information Displays, Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials, Jiangsu National Synergetic Innovation Centre for Advanced
Materials, Nanjing University of Posts and Telecommunications, Nanjing 210023, China. 3School of Physical and Mathematical Sciences, Nanjing Tech University
(NanjingTech), Nanjing 211800, China.
*Corresponding to: Heng Dong, Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Nanjing University, No.30 Zhongyang Road, Nanjing
210008, China. E-mail: dongheng90@smai l.nju.edu.cn.
Microbubbles (MBs) are gas-filled micrometer-scale spheres that are
commonly formed by the gas core encapsulated with stabilizing shells,
including polymers, surfactants, proteins, or liposomes shells.
Clinically, MBs were originally used as contrast agents for enhanced
ultrasound (US) imaging and diagnostics. Nowadays, MBs were given
expectations that they can be alternative platforms for drug delivery
owing to their unique acoustic properties. MBs can respond to the US
by cavitation effect which refers to a series of complex dynamic
processes, such as oscillation, expansion, contraction, and implosion
[1]. Drug molecules or therapeutic agents can be associated with the
MB shells by means of van-der-Waals forces, electrostatic or
hydrophobic interactions, or merely by physical encapsulation [2].
Therefore, strategies are emerging which take advantages of
US-mediated MBs drug delivery systems, mainly focusing on
sonothrombolysis, cancer therapy and central nervous system (CNS)
pathologies [3]. Nevertheless, several researchers have apperceived
the promising potential of US-responsive MBs in antibacterial therapy.
Here, we aimed to paint an overview of the latest published papers on
MBs for antibacterial therapy, hoping to help understand the
perspectives that the field may offer emerging generations of
antibacterial agents.
When assisted with the US, MBs not only play the role of drug
carriers, the cavitation effects of MBs can create pores in cell
membranes temporarily and lead to an efficient increase of cell
membrane permeability. This results in enhanced tissue distribution
and intracellular delivery of antibacterial agents, which is rather
important for breaking through biological barriers and treat some
obstinate infections [4]. For example, Horsley et al.developed novel
US-activated lipid MBs for urinary tract infection (UTI) [5]. During
acute UTI, the common uropathogenic bacteria can invade the
urothelial wall and form dormant reservoirs within cells where they
may escape luminal antibiotics and the immune system. This is one
possible explanation for high UTI recurrence rates after oral antibiotic
treatments which have difficulty in penetrating the bladder wall and
accumulating to an effective concentration. Therefore, Horsley and
co-workers developed US-activated sulfur hexafluoride-filled MBs
whose shells were constructed from lipids and decorated with
liposomes and gentamicin. Confocal results showed that US-activated
intracellular delivery of MBs in the human urothelial organoid model
was over 16 times greater than the control group and double that of
liposomes without MBs. The authors then infected human urothelial
organoids with a patient-isolated strain of E. faecalis, and the results
confirmed the effectiveness of killing and clearing uropathogenic
bacteria by US-activated microbubble therapy. Besides intracellular
infection like UTIs, some CNS infections also challenge traditional
antibacterial therapy and threaten human life in a clinic because of
the blood-brain barrier (BBB). BBB is established by the neurovascular
unit (NVU), which helps to regulate influx and efflux transport, thus
maintaining brain homeostasis and protecting the brain from harm
[6]. On the other hand, BBB limits the transport of most therapeutic
agents and entities into CNS when neurological diseases happen [7].
Therefore, research for blood-brain-barrier disruption (BBBD)
methods is crucial, whose goal is to compromise the BBB transiently to
allow the circulating therapeutic molecules and biomarkers to pass
into the parenchyma and, less frequently, out of it [8]. Focused-US
with MBs is an attractive noninvasive approach for permeabilizing the
BBB, owing to its adjustable and transient impact on the vasculature,
as well as a significant number of tunable parameters that can
influence its safety and efficacy. Xu et al.prepared a
tigecycline-loaded, US-activated MBs for the treatment of CNS
infectious disease caused by the multidrug-resistant Acinetobacter
baumannii (AB) [9]. The results indicated that the US improves the
anti-AB performance of tigecycline-loaded MBs in vitro. The authors
expected to apply pulsed US in conjunction with the intravenous
infusion of tigecycline-loaded MBs to treat intracranial AB infections
in animals. While the authors have not published the work yet, it is
worth paying attention to subsequent progress. In conclusion,
US-activated MBs may have great potential in crossing biological
barriers for the treatment of infectious diseases, including but not
limited to cell walls and BBB.
The above-mentioned studies are all about the treatment for
planktonically growing, suspended bacteria, while bacterial infections
are mostly caused by bacteria in an adhering and biofilm mode of
growth [10]. Biofilms are structured communities of microbial cells
where bacteria are embedded in the matrix, which is composed of
extracellular polymeric substances (EPS) [11, 12]. EPS functions as
physical barrier to enable bacteria within biofilm to escape the host
immune response and treatment of anti-bacterial agents [13].
Therefore, it is important to destroy the biofilm structural barrier
which protects the bacteria [14]. US-assisted microbubble therapy
may perform well due to its ability to break through barriers. To be
specific, under US stimulation, MBs can contract in an oscillatory
manner and generate local micro-streams by cavitation, which can
disrupt the structures of biofilms and produce holes and channels
within biofilms to enhance the penetration of drug molecules [15].
Kouijzer et al.decorated lipid MBs with vancomycin as a therapeutic
agent against Staphylococcus aureus biofilms [16]. The authors
confirmed the microbubble oscillation and biofilm disruption upon
ultrasound exposure using time-lapse confocal microscopy combined
with the ultra-high-speed camera. Results showed that upon US in
sonification, biofilm area was reduced by up to 28%. Moreover,
microbubbles can eliminate biofilms not only by physical destruction
and the release of antibiotics but also by chemical degradation and the
activation of the host immune response for pathogen clearance when
MBs are well-designed. Xiu et al.designed self-assembly microbubbles
composed of Fe3O4nanoparticles (NPs) loading piperacillin (MB-Pip)
to enhance biofilm elimination and immune activation to treat chronic
lung infections (Figure 1) [15]. Besides the physical disruption of
Pseudomonas aeruginosa biofilms and penetration of Pip, the released
Fe3O4NPs with peroxidase-like catalytic activity can catalyze H2O2to
generate hydroxyl radicals (•OH) and chemically degrade the biofilm
matrix. Also, both in vitro and in vivo results indicated that the Fe3O4
NPs released from MB-Pip can induce proinflammatory macrophage
(M1-like phenotype) infiltration and polarization which improved the
aberrant state of the host inflammatory response. In addition, some
studies have found that perfluoro pentane (PFP) or perfluorocarbon
(PFC) liquid can be made as nanodroplets and ultrasound-induced
pressure variations can cause them to vaporize due to the low boiling

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point of PFP or PFC, thereby converting these droplets into gas
bubbles [4, 17, 18]. Compared to pre-prepared MBs, those
transformed from microdroplets are more stable and controllable and
possess a longer half-life in vivo [19]. Xin et al.developed such
US-activatable phase-shift microdroplets by a double emulsion
approach, where the PFP and antibiotic meropenem (MEM) were
encapsulated within poly (lactic-co-glycolic acid) (PLGA) NPs [20].
Also, to target and eradicate Pseudomonas aeruginosa (P. aeruginosa)
biofilms, the surface was conjugated to a P. aeruginosa-specific
monoclonal antibodies via 1-(3-(dimethylamino)propyl)-3-ethyl
carbodiimide hydrochloride/N-hydroxysuccinimide (NHS/EDC)
chemistry. Results demonstrated a satisfactory antibiofilm effect when
microbubbles were triggered by the US. And no changes in zeta
potential and dynamic light scattering (DLS) analyses for
microdroplets stored in PBS over a 21 days period indicated great
stability under physiological conditions. Overall, MBs present a
promising sonobactericidal approach to biofilm elimination, thereby
fighting against infections and relieving inflammation.
Figure 1 Schematic illustration of US-activated MBs for efficient treatment of chronic lung infections by promoting biofilm elimination
and immune activation
Above all, US-activated microbubble therapy could be considered a
promising method for bacterial infection. However, there are still
many aspects that need to be studied and figured out. The
formulations, size, composition, dose and pharmacokinetics of MBs, as
well as ultrasound parameters, matter to effective and safe
antibacterial behavior in different kinds of infectious diseases. For
example, the balance between treatment effectiveness and BBBD
safety has been a long-standing concern when it comes to the
standardization of parameters for ultrasound and microbubbles [8].
MBs may induce leakage of the BBB vasculature and cause significant
adverse effects like hemorrhages, sterile inflammation or
neurotoxicity [21]. In summary, additional technical knowledge and
fundamental research will be essential to allow the evolution of
therapeutic US-activated MBs into a safe technique for antibacterial
applications.
References
1. Wu J, Nyborg WL. Ultrasound, cavitation bubbles and their
interaction with cells. Adv Drug Delivery Rev
2008;60(10):1103–1116. Available at:
http://doi.org/10.1016/j.addr.2008.03.009
2. Tinkov S, Bekeredjian R, Winter G, Coester C. Microbubbles as
ultrasound triggered drug carriers. J Pharm Sci
2009;98(6):1935–1961. Available at:
http://doi.org/10.1002/jps.21571
3. Fournier L, de La Taille T, Chauvierre C. Microbubbles for
human diagnosis and therapy. Biomaterials 2023;294:122025.
Available at:
http://doi.org/10.1016/j.biomaterials.2023.122025
4. Chowdhury SM, Abou-Elkacem L, Lee T, Dahl J, Lutz AM.
Ultrasound and microbubble mediated therapeutic delivery:
Underlying mechanisms and future outlook. J Controlled Release
2020;326:75–90. Available at:
http://doi.org/10.1016/j.jconrel.2020.06.008
5. Horsley H, Owen J, Browning R, et al. Ultrasound-activated
microbubbles as a novel intracellular drug delivery system for
urinary tract infection. J Controlled Release 2019;301:166–175.
Available at:
http://doi.org/10.1016/j.jconrel.2019.03.017
6. Obermeier B, Daneman R, Ransohoff RM. Development,
maintenance and disruption of the blood-brain barrier. Nat Med
2013;19(12):1584–1596. Available at:
http://doi.org/10.1038/nm.3407
7. Wang J, Li Z, Pan M, et al.Ultrasound-mediated blood-brain
barrier opening: An effective drug delivery system for
theranostics of brain diseases. Adv Drug Delivery Rev
2022;190:114539. Available at:
http://doi.org/10.1016/j.addr.2022.114539
8. Song KH, Harvey BK, Borden MA. State-of-the-art of
microbubble-assisted blood-brain barrier disruption.
Theranostics 2018;8(16):4393–4408. Available at:
http://doi.org/10.7150/thno.26869
9. Xu Y, Ren Y, Zhu Y, et al. Preparation, characterization, and
antibacterial activity of tigecycline-loaded,
ultrasound-activated microbubbles. Pharm Dev Technol
2021;27(1):1–8. Available at:
http://doi.org/10.1080/10837450.2021.2017967
10. Liu Y, Shi L, Su L, et al. Nanotechnology-based antimicrobials
and delivery systems for biofilm-infection control. Chem Soc Rev
2019;48(2):428–446. Available at:
http://doi.org/10.1039/C7CS00807D
11. Hu Y, Ruan X, Lv X, et al.Biofilm microenvironment-responsive
nanoparticles for the treatment of bacterial infection. Nano
Today 2022;46:101602. Available at:
http://doi.org/10.1016/j.nantod.2022.101602
12. Lv X, Wang L, Mei A, et al.Recent Nanotechnologies to
Overcome the Bacterial Biofilm Matrix Barriers. Small
2022;19(6):2206220. Available at:
http://doi.org/10.1002/smll.202206220
13. Karygianni L, Ren Z, Koo H, Thurnheer T. Biofilm Matrixome:
Extracellular Components in Structured Microbial Communities.
Trends Microbiol 2020;28(8):668–681. Available at:
http://doi.org/10.1016/j.tim.2020.03.016
14. Dong H, Xiu W, Wan L, et al. Biofilm microenvironment
response nanoplatform synergistically degrades biofilm
structure and relieves hypoxia for efficient sonodynamic
therapy. Chem Eng J 2023;453:139839. Available at:

COMMENT
Biomedical Engineering Communications 2023;2(2):7. https://doi.org/10.53388/BMEC2023007
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Submit a manuscript: https://www.tmrjournals.com/bmec
http://doi.org/10.1016/j.cej.2022.139839
15. Xiu W, Ren L, Xiao H, et al.Ultrasound-responsive catalytic
microbubbles enhance biofilm elimination and immune
activation to treat chronic lung infections. Sci Adv
2023;9(4):eade5446. Available at:
http://doi.org/10.1126/sciadv.ade5446
16. Kouijzer JJP, Lattwein KR, Beekers I, et al.
Vancomycin-decorated microbubbles as a theranostic agent for
Staphylococcus aureus biofilms. Int J Pharm 2021;609:121154.
Available at:
http://doi.org/10.1016/j.ijpharm.2021.121154
17. Phillips LC, Puett C, Sheeran PS, Dayton PA, Wilson Miller G,
Matsunaga TO. Phase-shift perfluorocarbon agents enhance
high intensity focused ultrasound thermal delivery with
reduced near-field heating. J Acoust Soc Am
2013;134(2):1473–1482. Available at:
http://doi.org/10.1121/1.4812866
18. Fernandes DA, Fernandes DD, Li Y, et al.Synthesis of Stable
Multifunctional Perfluorocarbon Nanoemulsions for Cancer
Therapy and Imaging. Langmuir 2016;32(42):10870–10880.
Available at:
http://doi.org/10.1021/acs.langmuir.6b01867
19. Sridharan A, Eisenbrey JR, Forsberg F, Lorenz N, Steffgen L,
Ntoulia A. Ultrasound contrast agents: microbubbles made
simple for the pediatric radiologist. Pediatr Radiol
2021;51(12):2117–2127. Available at:
http://doi.org/10.1007/s00247-021-05080-1
20. Xin L, Zhang C, Chen J, et al.Ultrasound-Activatable
Phase-Shift Nanoparticle as a Targeting Antibacterial Agent for
Efficient Eradication of Pseudomonas aeruginosa Biofilms. ACS
Appl Mater Interfaces 2022;14(42):47420–47431. Available at:
http://doi.org/10.1021/acsami.2c13166
21. Deprez J, Lajoinie G, Engelen Y, De Smedt SC, Lentacker I.
Opening doors with ultrasound and microbubbles: Beating
biological barriers to promote drug delivery. Adv Drug Delivery
Rev 2021;172:9–36. Available at:
http://doi.org/10.1016/j.addr.2021.02.015
Author contribution
Xiaoye Li wrote the draft and drew the figure. Dongliang Yang collected relevant
information. Weijun Xiu and Heng Dong edited the text and finalized the manuscript. All
authors have reviewed and agreed to the final draft.
Competing interests
The authors declare no conflicts of interest.
Acknowledgments
This work was financially supported by “3456” Cultivation P rogram for Junior Talents of
Nanjing Stomatological Hospital, Medical School of Nanjing University (No. 0222R212),
Natural Science Foundation of Jiangsu Province (No. BK20200710).
Abbreviations
MBs, microbubbles; US, ultrasound; CNS, central nervous system; UTI, urinary tract
infection; BBB, blood-brain barrier; NVU, neurovascular unit; BBBD, blood-brain-barrier
disruption; EPS, extracellular polymeric substances; NPs, nanoparticles; MB-Pip,
microbubbles composed of Fe3O4nanoparticles loading piperacillin; •OH, hydroxyl
radicals; PFP, perfluoropentane; PFC, perfluorocarbon; PLGA, poly(lactic-co-glycolic acid);
EDC, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride; NHS,
N-hydroxysuccinimide; DLS, dynamic light scattering.
Citation
Li XY, Xiu WJ, Yang DL, Dong H. Ultrasound-responsive microbubbles in antibacterial
therapy. Biomed Eng Commun. 2023;2(2):7. doi: 10.53388/BMEC2023007.
Executive editor: Na Liu.
Received: 21 April 2023, Accepted: 25 April 2023, Available online: 28 April 2023.
© 2023 By Author(s). Published by TMR Publishing Group Limited. This is an open
access article under the CC-BY license. (https://creativecommons.org/licenses/by/4.0/).