Advances of therapeutic microbubbles and nanobubbles

: As versatile nanotools, microbubbles (MBs) and nanobubbles (NBs) have gained increasing attention because of their great potentials for therapeutic applications. The filled gases can greatly influence the physical properties of the MBs and NBs, such as size, zeta potential, and stability. Importantly, the biological activity of the filled gases plays a key role in the therapeutic effect of MBs and NBs. In this review, we will introduce the background, classification, and design strategies of therapeutic MBs and NBs based on bio-active and bio-inactive gases. Furthermore, focus will be placed on the biomedical applications in cancer treatments, including drug delivery, targeting effect and combinations of different therapeutic modalities. Finally, we will discuss the remaining challenges and future perspectives of therapeutic MBs and NBs.


Introduction
With the rapid development of nanotechnology, various nanoplatforms such as nanomedicine and nanodelivery systems have been explored and developed for the therapy of diseases in clinic over the past few decades [1][2][3][4][5][6].Among them, gaseous systems including micro-and nano-sized bubbles with spherical gasfilled and core-shell structures have gained widespread attention because of their distinct advantages, such as flexibility of compositions and structures, favorable and controllable sizes, desirable biocompatibility, high surface area, enhanced permeability and retention, passive targeting and accumulation in lesion region [7][8][9][10].Generally, microbubbles (MBs) have a diameter of more than 1 μm, which is approximately equal to the diameter of a red blood cell.The large size of MBs limits their extravasation from the bloodstream.MBs are traditionally served as ideal ultrasound (US) contrast agents (UCAs) for imaging because of their frequency response to US waves.In medical fields, MBs can also serve as efficient carriers of drugs, gases, genes, etc.Recent studies have shown that the assembled MBs can be transformed into nanoparticles upon US to improve the therapy effect [3].In contrast, the diameter of nanobubbles (NBs) are decreased to 100 nm-1 μm.Due to the small size, NBs can extravasate from leaky bloodstream into tumor tissues, indicating great potentials for therapeutic applications.Different gases contained in MBs and NBs have an impact on the size, zeta potential, stability, and therapeutic effect.According to the biological activity of the filled gases, MBs and NBs can be classified into two major types (Figure 1): (1) bio-active gases based MBs and NBs; (2) bio-inactive gases based MBs and NBs.
Bio-active gases based MBs and NBs.Some gases such as oxygen (O 2 ), nitric oxide (NO), xenon (Xe), carbon monoxide, hydrogen and hydrogen sulfide are demonstrated to affect various physiological and pathophysiological processes and involve in some biological activities [3].For instance, O 2 is well known for respiration to sustain life.Due to the inadequate supply of O 2 to the tissues, hypoxia may occur and lead to various implications.Hypoxia is considered to be the major cause inducing the invasion and metastasis of solid tumor and this phenomenon may restrict the therapeutic effect of chemotherapy, radiotherapy, sonodynamic therapy (SDT), and photodynamic therapy (PDT).O 2 MBs and NBs may provide a facile method to satisfy the urgent need for hypoxia-overcoming treatments [11][12][13][14][15][16][17][18][19][20].As a biological signaling molecule, NO acts as an important regulator in many physiological processes, such as protecting against ischemia-reperfusion injury [21], anti-inflammatory and immunosuppressive effects, reducing platelet aggregation and thrombosis [22], and promoting endothelium regeneration for regulation of the cardiovascular system [23].However, the therapeutic application of NO depends on its concentration in vivo.High concentration (>1 mmol/L) NO in the blood can cause poisoning, while low concentration (10 −12 to 10 −9 mol/L) NO in cancer cells may promote cancer cell growth.It is important to achieve controllable delivery and release of NO for clinical translation.NO MBs are born on demand and applied in the treatments of some diseases [24][25][26][27][28][29][30][31].As a noble gas, Xe is able to traverse the blood-brain barrier due to its small volume and low blood gas partition coefficient [32].In various hypoxic-ischemic injury cases, Xe is a bioactive gas that has been Natl Sci Open, 2023, Vol.2, 20220062 demonstrated to have anti-apoptotic effects [33].Xe also shows neuroprotective properties by inhibiting the N-methyl-D-aspartate without coexisting neurotoxicity [34].In addition, Xe has cardioprotective [35] and renoprotective effects [36] in vivo.Due to Xe's scarcity and high cost, the primary problem of Xe therapy lies in effective delivery and release of Xe.Liposomes and phospholipid are applied to encapsulate Xe MBs or NBs for the localized delivery of Xe and treatments of some diseases [37][38][39][40].Up to now, due to instability or insecurity, other bioactive gases such as carbon monoxide, hydrogen and hydrogen sulfide based MBs and NBs are seldom reported and applied as therapeutic bubbles.
This review will first introduce the background and classification of therapeutic MBs and NBs.Subsequently, the design strategies of therapeutic MBs and NBs will be summarized.Especially, emphasis will be put on the therapeutic applications of MBs and NBs (Table 1), including drug delivery, targeting effect and combinations of different therapeutic modalities.In the end, the challenges and prospects of therapeutic MBs and NBs will be discussed.

Bio-active microbubbles and nanobubbles Oxygen microbubbles and nanobubbles
Figure 2 illustrates the design strategies of O 2 MBs and NBs for biomedical applications.Hypoxia is a vital factor that may restrict the therapeutic effect of solid tumors, and hypoxia-inducible factor 1-α (HIF-1α) is known as an important regulator of O 2 homeostasis and hypoxia adaptation in tumour cells.With the accumulation of HIF-1α, tumour cells may acquire resistance to radiation under hypoxic conditions.Iijima et al. [13] demonstrated that the use of O 2 NBs water could overcome the hypoxia-induced resistance via inhibiting the expression of HIF-1α.In this report, stable O 2 NBs with sizes ranging from 2 to 3 nm were fabricated using the ΣPM-5 device.In contrast to the cells (EBC-1 lung tumour and MDA-MB-231 breast tumour cells) grown in medium consisting of distilled water, the tumour cells grown in medium made with O 2 NBs-containing water showed a clear suppression of HIF-1α expression.
Khan and coworkers [14]   Indocyanine green (ICG), a clinically near-infrared fluorophore approved by U.S. Food and Drug Administration [19], can serve as a photosensitizer for PDT of tumours.However, a major hurdle of ICG in PDT lies in its poor singlet oxygen ( 1 O 2 ) production and instability in aqueous solution.To solve these issues, our group [20] developed ICG-NBs-O 2 , in which ICG molecules are assembled with O 2 NBs by hydrophilichydrophobic interactions.Compared with free ICG, ICG-NBs-O 2 exhibits better stability in solution.Meanwhile, the 1 O 2 production of ICG-NBs-O 2 is significantly increased to be 1.6%, which is eight times as high as that of free ICG (0.2%).Furthermore, ICG-NBs-O 2 shows enhanced PDT in the treatment of oral tumours.

Nitric oxide microbubbles
NO can inhibit platelet aggregation by providing an inactivating signal to the protein membrane integrins, thereby controlling arterial thrombosis and cardiovascular disease (Figure 3).However, sometimes the natural supply of NO is insufficient to inhibit blood clotting.Therefore, it is necessary to provide exogenous NO for efficient therapy.Cavalieri et al. [24] designed the NO-loaded poly(vinyl alcohol) shelled MBs and investigated the loading capacity and delivery of NO.The results indicate that NO MBs are effective to transport and release NO as anticlotting agent in vitro.
NO MBs are also fabricated and used for thrombi treatment [21].By loading with MBs, the circulation time of NO in vivo can be remarkably improved.Moreover, the NO can be released effectively under US radiation.The results show that the thrombus area is significantly reduced and the recanalization rates and blood flow velocities are improved compared with the control group.
High levels of NO have been proved to play a key role in the immune system, mainly through antiinflammatory and immunosuppressive effects [25].It is reported that NO can effectively inhibit not only macrophage activation but also the proliferation and recruitment of T and B cells [26,27].Liao et al. [22] developed C4d-targeted NO MBs and used for antibody-mediated rejection studies.Non-invasive diagnosis of antibody-mediated rejection is achieved by quantitative analysis of contrast-enhanced US images after C4d-targeted NO MBs injection.In addition, allograft survival is significantly prolonged and rejection is significantly reduced by inhibiting thrombus formation and reducing inflammatory cell infiltration.Therefore, C4d-targeted NO MBs have shown significantly increased therapeutic efficacy compared with the control group.
Due to the important role of NO in cardiovascular homeostasis, NO MBs have also been studied in the treatment of myocardial infarction [28].By investigating the effects of US combined with the destruction of NO MBs on the fate of mesenchymal stem cells, it is found that mesenchymal stem cells migrate more efficiently in the group treated with NO MBs under US radiation.Furthermore, the results of the research on the effect of NO MBs on bone marrow mesenchymal stem cells infusion show that NO MBs can be used for cell transplantation and effectively promote the homing of mesenchymal stem cells to the infarcted myocardium upon US irradiation.NO has been reported to partially mediate signaling in the early stages of glucose-stimulated insulin secretion [29].However, the delivery and manipulation of NO to biological systems is still relatively difficult because of the reactive chemical nature of NO.Based on glucose and magnetic response, our group [30] developed a theranostic delivery system to generate NO MBs by reacting H 2 O 2 with L-arginine in situ, which can be used to modulate glucose hemostasis and spatiotemporally control NO release and delivery.On db/db type 2 diabetic mouse model, NO therapy for renal hypoxia is achieved by continuous generation of NO in situ.In further work, our group [31] fabricated a nanocarrier by loading L-arginine and γ-Fe 2 O 3 magnetic nanoparticles on a natural platelet membrane for targeted delivery of L-arginine and generation of NO in situ.Upon the stimulus of magnetic field, the nanocarriers can target ischemic stroke lesions, release L-arginine, and produce NO in situ.As a result, generation of NO in situ enhances vasodilation and reduces platelet aggregation, which can be used for the therapy of early ischemic stroke.

Xenon microbubbles and nanobubbles
Xe MBs and NBs have great potentials in therapeutic applications (Figure 4).Irani et al. [38] fabricated lipidshelled MBs loaded with Xe by high shear mixing method.The release of gaseous payloads can be triggered using Doppler and pulsed US, and these results suggest the potential application of lipid-shelled MBs in stroke neuroprotection.After intravenous injection of Xe MBs, an US pulse is applied to the carotid artery to release Xe from the MBs into the brain [39].Based on a high-fidelity pig model, the neuroprotective efficacy of Xe MBs in traumatic brain injury is confirmed.
Because of its ability to diffuse across the blood-brain barrier and safety through expiration, Xe has unique therapeutic advantages in the treatment of ischemic stroke.Recently, our group [35] used phospholipid membrane to encapsulate Xe to construct NBs with sizes of 225 ± 11 nm.Xe NBs are applied to treat acute Natl Sci Open, 2023, Vol.2, 20220062 ischemic stroke with neuroprotection and microcirculation restoration.The biodistribution of Xe NBs in mice with ischemic stroke suggests that the enhanced accumulation of Xe NBs in cerebral ischemic lesions may enable contrast-enhanced US imaging with lesion areas and effective delivery of Xe.Compared with the control group, the efficacy of Xe NBs in the treatment of stroke is improved, which provides a new strategy for the therapy of acute ischemic stroke.

Perfluorocarbon microbubbles and nanobubbles
PFC MBs and NBs attract increasing attention in the construction of efficient delivery system.To improve the delivery efficacy of doxorubicin and reduce the side effects, doxorubicin-loaded C 3 F 8 -cored glycol chitosan NBs are designed and fabricated [41].With the combination of chemotherapy and extracorporeal shock waves under US irradiation, doxorubicin-loaded C 3 F 8 NBs exhibit enhanced anti-tumor effects and decreased heart damage.Aimed to obtain efficient theranostic agent for SDT, a series of porphyrin derivatives are loaded on C 3 F 8 NBs by manipulating the hydrophilic/lipophilic balance [42].Under US irradiation, the optimized NBs system exploits the ability to produce ROS and shows a significant reduction in cell viability of LS 174T colorectal cancer.
To conquer multidrug resistance in the chemotherapy of colorectal tumour, Dai's group [43] developed a multimodal therapeutic agent PCF-MBs with high drug loading capacity (w/w: porphyrin 4.5%, floxuridine 14.3% and camptothecin 20.2%) and excellent drug delivery ability.Interestingly, PCF-MBs with sizes of about 1 μm can be converted into nanoparticles with sizes of 30 to 100 nm.Due to the combination of US, chemotherapy and PDT, PCF-MBs can not only significantly reduce the ABCG 2 expression associated with the drug resistance in chemotherapy but also achieve great inhibition of HT-29 colorectal cancer.
Sun et al. [44] designed and synthesized multifunctional MBs, siHIF@CpMBs, which contain cationic porphyrin-grafted lipid as a photosensitizer, HIF 1α siRNA as a gene therapy agent for down-regulating the level of HIF 1α and C 3 F 8 MBs.siHIF@CpMBs shows a high loading capacity of 24.2% (porphyrin, W/W%) and efficient adsorption of HIF 1α siRNA, indicating its PDT and gene therapy potential.With the destruction of MBs upon US treatment, siHIF@CpMBs is efficiently converted into nanoparticles, leading to the efficient accumulation of porphyrin and siRNA at tumour sites.The results show that knockdown of HIF 1α expression via HIF 1α siRNA can enhance the PDT efficacy and partly inhibit the tumour progression.
To enhance SDT treatment, a Rose Bengal derivative sonosensitizer is covalently attached to the surface of C 4 F 10 MBs.Upon US irradiation, the resulting conjugate shows increased singlet oxygen production and significantly reduces the tumor growth in LNCaP-Luc prostate cancer model [45].To improve the generation of ROS and delivery efficiency of sonosensitizers in SDT, rose bengal MBs (RB-MBs-C 3 F 8 ) are designed and fabricated by encapsulating C 3 F 8 MBs with amphiphilic rose bengal.RB-MBs-C 3 F 8 exhibits high drugloading capability (~6.8%) and good contrast enhancement ability for US imaging, simultaneously.Similar to the PCF-MBs mentioned above, RB-MBs-C 3 F 8 can also be transformed to nanoparticles under US irradiation.Compared with the corresponding RB-MBs-C 3 F 8 without US irradiation, US-activated RB-MBs-C 3 F 8 shows significantly higher drug accumulation and tumour inhibition in HT-29 colorectal cancer model [46].
As a promising strategy, physical non-drug therapy has attracted increasing attention.Based on folic acid modified N-palmitoylc hitosan and C 3 F 8 NBs, a novel FNBs-C 3 F 8 is designed and fabricated.FNBs-C 3 F 8 can effectively target folate receptor-positive tumour cells.Interestingly, FNBs-C 3 F 8 can kill folate receptorpositive tumour cells through intracellular explosion upon therapeutic US irradiation [47].
Molecule-targeted UCAs are desirable for accurate diagnosis and targeted therapy of cancer.Multifunctional UCAs based on NBs have attracted increased attention.Shen et al. [48] fabricated multifunctional NBs, FA-NBs-IR780, in which a targeting agent folic acid and a near-infrared fluorescence dye IR-780 iodide are assembled on lipid C 3 F 8 NBs, respectively.Due to the dual targeting ability, FA-NBs-IR780 can efficiently target tumour cells as expected.More importantly, FA-NBs-IR780 shows striking photothermal responsiveness under 808 nm irradiation and can be used as a photothermal agent for efficient PTT.

Sulfur hexafluoride microbubbles and nanobubbles
To improve the stability of UCAs, people tend to other gases with poor solubility in water.Schneider et al. [51] developed BR1, a suspension of SF 6 MBs for efficient UCAs.The MBs in BR1 have a mean diameter of 2.5 μm, a favourable size distribution and high bubbles concentration of 2 × 10 8 mL −1 , resulting in highly echogenic suspensions.Compared with surface NBs, production of stable bulk NBs is still at the initial stage.Recently, our research group [52] developed the repeated compression method for the fabrication of bulk SF 6 NBs.The average particle size of the generated SF 6 NBs is 240 nm and the polydispersity coefficient is 0.25.The SF 6 NBs have a high negative zeta potential with good stability of more than 48 h.Additionally, the formation mechanism of SF 6 NBs was investigated using dark-field microscopy [53].The dynamic evolution of bulk NBs in situ is visualized and traced with dark-field microscopy, and it is directly observed that NBs are formed by the shrinkage of MBs.A comparative study shows that MBs carrying different types of gases (Xe, air, and SF 6 ) exhibit different shrinkage rates due to their different gas barrier properties.However, the final stable NBs do not differ significantly in sizes and zeta potential.Further, the bubble stabilization mechanism is investigated by the attenuated total reflection Fourier transform infrared spectroscopy.According to the experimental results, it is speculated that the strong hydrogen bonding at the air-water interface may be responsible for promoting the stability of NBs.SF 6 NBs show excellent potentials in the development of therapeutic nanomaterials (Figure 5).To overcome poor water stability and concentration-dependent aggregation of ICG mentioned above, our group [54] designed and fabricated ICG-NBs-SF 6 by self-assembly of ICG with SF 6 NBs.Compared with free ICG solution, ICG-NBs-SF 6 exhibits increased fluorescence intensity and stability.Very recently, our group [55] prepared SF 6 NBs coated with a high concentration of superparamagnetic iron oxide nanoparticles.US and magnetic resonance imaging experiments show that the assembled magnetic NBs exhibited good contrast between US and magnetic resonance images.In addition, the magnetic NBs are used to label neural stem cells under US excitation.Compared with the ordinary incubation method, the US irradiation method is about twice as efficient as the incubation method.
Platelet membranes have been used in the development of biomimetic diagnostic and therapeutic nanomaterials due to their good biocompatibility and effectiveness.Our group [56] prepared platelet membraneencapsulated SF 6 NBs with ideal echogenicity by a tunable pressure-induced shear stress method.The results show that high shear stress during the fabrication of platelet membrane-encapsulated SF 6 NBs enables the enrichment of platelet membrane lipid rafts and proteins and their assembly at the air-liquid interface of SF 6 Natl Sci Open, 2023, Vol.2, 20220062 free bubbles.Due to the conformation shift of platelet integrin αIIbβ3 to a shear stress-induced intermediateaffinity state, platelet membrane-encapsulated SF 6 NBs enhance the ability to adhere to vascular endothelial injury and show great application potential in the specific targeted US diagnosis of vascular endothelial injury.
Neural stem cells (NSCs) have been used in the treatment of various neurological diseases because of their self-renewal ability and multi-directional differentiation potential.However, the tracking of migratory capacity in vivo and the control of survival and differentiation efficiency of NSCs are insufficient, limiting the clinical application.To modulate NSCs fate, our group designed and fabricated magnetic NBs-labeled NSCs, in which magnetic NBs assembled from magnetic nanoparticles and SF 6 NBs are internalized by NSCs.In the case of magnetic SF 6 NBs labeled NSCs, intramembrane volume oscillations of magnetic NBs-labeled NSCs may induce increases in intracellular hydrostatic pressure and cytoskeletal force, resulting in Piezo1-Ca 2+ machinery sensory channel activation.This in turn triggers the BMP2/Smad biochemical signaling pathway, leading to NSCs differentiation into a neuronal phenotype.Signaling through the Piezol-Ca 2+ -BMP2/Smad pathway can be further accelerated using low-intensity pulsed US.Therefore, it is possible to utilize magnetic NBs-labeled NSCs for efficient NSCs treatment outcomes monitored by magnetic resonance and US imaging [57].
As a non-viral strategy, SF 6 MBs and NBs can be applied to deliver genes with the mediation of US [59].With the continuous advancement of gene editing technology, combining the CRISPR/Cas9 system with nanocarrier technology can improve the delivery efficiency and biocompatibility of genetic materials.Ryu et al. [58] designed SF 6 MBs conjugated nanoliposome system as a Cas9/sgRNA riboprotein complex carrier, which can effectively facilitate local delivery to specific sites under US irradiation.Additionally, the SF 6 MBs conjugated nanoliposome particle can transfer protein complexes into hair follicle cells and exhibit high Natl Sci Open, 2023, Vol.2, 20220062 efficiency in editing target genes in vitro and in vivo.Therefore, US-activated SF 6 MBs conjugated nanoliposome particle vectors may help expand the platform for protein-based gene editing.

Conclusions and future perspective
As promising biomedical nanotools, therapeutic MBs and NBs have shown significant potential in clinic for their excellent and unique properties.Therapeutic MBs and NBs are fabricated by sonication, mulsion, mechanical agitation, laser ablation, and other methods [7,36,52].Compared with therapeutic gas, therapeutic MBs and NBs have measurable sizes and good stability [20].As mentioned above, some functional MBs and NBs show specific treatments and good efficiency in therapy.However, therapeutic MBs and NBs still have some restrictions on their clinical applications.Some essential issues need to be addressed for the broad applications of therapeutic MBs and NBs.
(1) In comparison to conventional gas therapy, the gases especially bio-active gases involved in therapeutic MBs and NBs remain limited in number.Thus, there is ample room for developing additional types of multifunctional MBs and NBs to expand their therapeutic application.To date, it is still challenging to expand therapeutic MBs and NBs to more bio-active gases through the improvement of materials, methodologies and technologies.Especially, with the breakthrough in molecular mechanism of hydrogen in the body [60], more efforts should put into the development of hydrogen based MBs and NBs for biomedical application.
(2) As efficient nano-delivery systems, the drug loading rate and targeting ability of MBs and NBs should be improved.In general, MBs show better frequency response properties upon US than NBs.In contrast, due to the smaller size for permeating into the vascular wall, NBs possess better passive-targeting ability than MBs.Therefore, more efforts should be made to precisely regulate the sizes of the bubbles for larger drug loading rate, better targeting ability and frequency response properties simultaneously.
(3) Although many therapeutic MBs and NBs have been designed and investigated in clinical treatments, only a limited number of MBs have received FDA approval for clinical application.Therefore, it is urgent to carry out comprehensive clinical evaluation on therapeutic MBs and NBs, which may help accelerate their translation into clinics and fulfill their therapeutic potentials.

Figure 1
Figure 1 Schematic illustration of therapeutic MBs and NBs for different biomedical applications.Therapeutic MBs and NBs can be classified into two major types: (i) bio-active gases (O 2 , NO, Xe, H 2 , etc.) based MBs and NBs; (ii) bio-inactive gases (N 2 , SF 6 , PTC, etc.) based MBs and NBs.Functional substrate such as sensitizer, drug, targeting agent and gene are introduced on the MBs and NBs for enhanced PTT, SDT, PDT, chemotherapy, targeted therapy, and gene therapy.
efficient delivery of O 2 to the vicinity of the tumour, the production of ROS is enhanced under US irradiation.For the SDT treatment of BxPc-3 tumour cells, the O 2 MBs conjugate can significantly enhance the therapeutic efficacy due to the combination of O 2 delivery and a US-responsive therapeutic sensitiser on O 2 MBs.Bhandari et al. [17] developed O 2 NBs with sizes of 100-200 nm in diameter by encapsulating O 2 inside sodium carboxymethyl cellulose based polymeric shell via a cross-linking step.Through the delivery of O 2 , significant hypermethylation can be achieved in promoter DNA region of BRCA1.The release of O 2 inside the hypoxic xenografted MB49 (bladder tumor) and HeLa (cervical tumor) cells may weaken the hypoxiamotivated pathways and suppress tumor growth.In addition, O 2 NBs can also reprogram several hypoxia associated and tumour suppressor genes such as MAT2A and PDK-1.This method is expected to have a significant impact on epigenetic programming and serve as an adjuvant to the treatment of tumours.Biogenic gas vesicles (GVs) are naturally formed in cynobacteria or archaea and made of protein with stable nanostructure (about 250 nm in diameter) [18].To improve the stability of O 2 NBs and enhance PDT of hypoxic tumours, Song and coworkers [18] developed O 2 -filled GVs as a carrier for effective O 2 delivery.With the addition of O 2 -filled GVs, O 2 concentration increased in hypoxic solution and cells, indicating that GVs can carry enough O 2 and have an effect on cell culture.In the presence of O 2 -filled GVs, significant enhancement of tumour cell apoptosis and necrosis was observed during PDT.

Figure 2
Figure 2 Schematic illustration of the design strategies of O 2 MBs and NBs for biomedical applications.O 2 MBs and NBs can suppress the expression of HIF-1α expression.Stable O 2 MBs and NBs are fabricated by encapsulating O 2 with shells (proteins, lipids or polymers) and functional substrate (photosensitizer, drug or gene).O 2 MBs and NBs can increase O 2 supply, improve O 2 delivery, overcome hypoxic conditions and enhance therapeutic efficacy.

Figure 3
Figure 3 Simplified diagram illustrating functional mechanisms of NO MBs for biomedical applications.NO MBs are stabilized by shells with functional substrates and effective to transport and release NO under US radiation.On the other hand, NO MBs are generated by reacting H 2 O 2 with L-arginine in situ.

Figure 4
Figure 4 Xe MBs and NBs have anti-apoptotic effects and neuroprotective, cardioprotective and renoprotective properties.More importantly, Xe NBs has unique therapeutic advantages in the treatment of ischemic stroke.

Figure 5
Figure 5 Mindmap of SF 6 MBs and NBs development.The research on SF 6 MBs and NBs mainly involves three aspects: (i) the formation and stability mechanism; (ii) imaging applications include magnetic resonance, fluorescence and ultrasound imaging; (iii) therapeutic applications such as vascular endothelial injury, neurological diseases, and gene therapy.

Table 1
fabricated lipid-shelled O 2 NBs using a sonication method and assessed the expression of HIF-1α under hypoxic conditions in vitro experiments on MDA-MB-231 breast tumour cells.In comparison to the control sample, O 2 NBs can supply more O 2 and be used to reverse hypoxia.The results indicate that HIF-1α expression is reduced after the introduction of O 2 NBs.In further work, Khan et al. [15] synthesized doxorubicin-loaded O 2 NBs (Dox-NBs-O 2 ) and evaluated the effectiveness of Dox-NBs-O 2 to MDA-MB-231 breast tumour and HeLa cervical tumour cells.They found that Dox-NBs-O 2 could not only down-regulate HIF-1α and enhance reactive oxygen species (ROS) generation, but also reduce the amount of doxorubicin.Under normal or hypoxic conditions, Dox-NBs-O Summary of therapeutic MBs and NBs for different biomedical applications [16]e verified to be more effective in tumor cells compared with commercially available liposomal Dox and free Dox.To enhance the SDT effect under hypoxic conditions, McEwan et al.[16]investigated an oxygen-carrying MBs conjugate composed of lipid O 2 MBs and a biotin functionalised Rose Bengal sensitizer.Due to the