A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan.

Immersion vaccination, albeit easier to administer than immunization by injection, sometimes has challenges with antigen uptake, resulting in sub-optimal protection. In this research, a new strategy to enhance antigen uptake of a heat-inactivated Vibrio harveyi vaccine in Asian seabass (Lates calcarifer) using oxygen nanobubble-enriched water (ONB) and positively charged chitosan (CS) was explored. Antigen uptake in fish gills was assessed, as was the antibody response and vaccine efficacy of four different combinations of vaccine with ONB and CS, and two control groups. Pre-mixing of ONB and CS before introducing the vaccine, referred to as (ONB + CS) + Vac, resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. The integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) within a vaccine trial experiment set out to further evaluate how survival rates post exposure to V. harveyi might be improved. Antibody responses were measured over 42 days, and vaccine efficacy was assessed through an experimental challenge with V. harveyi. The expression of immune-related genes IL1ß, TNFα, CD4, CD8, IgT and antibody levels were assessed at 1, 3, and 7-day(s) post challenge (dpc). The results revealed that antibody levels in the group (ONB + CS) + Vac were consistently higher than the other groups post immersion immunization and oral booster, along with elevated expression of immune-related genes after challenge with V. harveyi. Ultimately, this group demonstrated a significantly higher relative percent survival (RPS) of 63 % ± 10.5 %, showcasing the potential of the ONB-CS-Vac complex as a promising immersion vaccination strategy for enhancing antigen uptake, stimulating immunological responses, and improving survival of Asian seabass against vibriosis.

Anti-HER2 scFv-nCytc-Modified Lipid-Encapsulated Oxygen Nanobubbles Prepared with Bulk Nanobubble Water for Inducing Apoptosis and Improving Photodynamic Therapy.

Bulk nanobubbles fascinate scientists because of their stability over long periods of time and their ability to carry gases, leading to numerous potential applications. Considering the hypoxic tumor microenvironment and the advantages of bulk nanobubbles, lipid-encapsulated oxygen nanobubbles are prepared from free bulk oxygen nanobubbles in this study. The obtained carrier is then modified with a protein fused with the single-chain antibody of human epidermal growth factor receptor 2 (anti-HER2 scFv) and tandem-repeat cytochrome c (anti-HER2 scFv-nCytc) to enhance tumor targeting and induce tumor apoptosis. Copper phthalocyanine is used as the photosensitizer to demonstrate how the oxygen in the nanobubbles affects the efficiency of photodynamic therapy (PDT). The combination of anti-HER2 scFv-nCytc and PDT synergistically improves the therapeutic effect and alleviates hypoxia in tumors in vivo while causing little inflammatory response. Based on the findings, bulk nanobubble water shows promise in the targeted delivery of oxygen and can be combined with antibody therapy to enhance the efficiency of PDT.

Interactions between O2 Nanobubbles and the Pulmonary Surfactant in the Presence of Inhalation Medicines.

A dispersion of oxygen nanobubbles (O2-NBs) is an extraordinary gas-liquid colloidal system where spherical gas elements can be considered oxygen transport agents. Its conversion into inhalation aerosol by atomization with the use of nebulizers, while maintaining the properties of the dispersion, gives new opportunities for its applications and may be attractive as a new concept in treating lung diseases. The screening of O2-NBs interactions with lung fluids is particularly needed in view of an O2-NBs application as a promising aerosol drug carrier with the additional function of oxygen supplementation. The aim of the presented studies was to investigate the influence of O2-NBs dispersion combined with the selected inhalation drugs on the surface properties of two types of pulmonary surfactant models (lipid and lipid-protein model). The characteristics of the air-liquid interface were carried out under breathing-like conditions using two selected tensiometer systems: Langmuir-Wilhelmy trough and the oscillating droplet tensiometer. The results indicate that the presence of NBs has a minor effect on the dynamic characteristics of the air-liquid interface, which is the desired effect in the context of a potential use in inhalation therapies.

Sustainable restoration of anoxic freshwater using environmentally-compatible oxygen-carrying biochar: Performance and mechanisms.

The long-term decline in dissolved oxygen (DO) levels in freshwater systems including rivers and lakes has become a worldwide concern, which can threaten biodiversity, nutrient biogeochemistry, water quality and ultimately human health. Herein, we report a sustainable restoration strategy for anoxic freshwater using local sediment-based biochar as novel oxygen nanobubble carriers. Column incubation experiments were conducted with water and sediment samples from an urban tributary of the Yangtze River. The oxygen-carrying sediment-based biochar (O-SBC) showed long-lasting re-oxygenation performance for anoxic river waters during 28-day period, in which DO was rapidly elevated from ∼0.14 to ∼7.87 mg/L and gradually maintained at ∼4.78 mg/L until the end. O-SBC with multiple functions switched the sediments from a source to a sink of nutrients, and its release of oxygen nanobubbles contributed further decrements of 66.3% NH4+-N and 142.9% PO43--P except for physical blocking and physicochemical adsorption. Notably, a comprehensive focus on restoration mechanism was explored in view of microbial community response. The re-oxygenation was followed by a ∼5.05% increase of bacterial diversity (Shannon index) in water, but a ∼2.40% decrease in sediments. A proliferation of some specific aerobic populations was observed, of which the nitrifying Nitrospira abundances were ∼10-fold higher in the water from O-SBC than the control. Additionally, functional genes involved in nitrous oxide reduction, polyphosphate synthesis/degradation, and thiosulfate oxidation were also enriched. Taken together, our findings can not only expand the promising candidates for oxygen nanobubble carriers based on sediment recycling, but also highlight the microbial molecular mechanisms for anoxic freshwater restoration based on nutrient cycle regulation.

Indocyanine green assembled free oxygen-nanobubbles towards enhanced near-infrared induced photodynamic therapy.

Photodynamic therapy (PDT) has shown a promising capability for cancer treatment with minimal side effects. Indocyanine green (ICG), the only clinically approved near-infrared (NIR) fluorophore, has been used as a photosensitizer for PDT in clinical application. However, the main obstacle of directly utilizing ICG in the clinic lies in its low singlet oxygen (1O2) quantum yield (QY) and instability in aqueous solution. To improve the PDT efficacy of ICG, free ICG molecules were assembled with free oxygen nanobubbles (NBs-O2) to fabricate ICG-NBs-O2 by hydrophilic-hydrophobe interactions on the gas-liquid interface. Interestingly, 1O2 QY of ICG-NBs-O2 solution was significantly increased to 1.6%, which was estimated to be 8 times as high as that of free ICG solution. Meanwhile, ICG-NBs-O2 exhibited better aqueous solution stability compared with free ICG. Furthermore, through establishing tumor models in nude mice, the therapeutic efficacy of ICG-NBs-O2 was also assessed in the PDT treatment of oral cancer. The tumor volume in ICG-NBs-O2 treated group on day 14 decreased to 0.56 of the initial tumor size on day 1, while the tumor volume in free ICG treated group increased to 2.4 times. The results demonstrated that ICG-NBs-O2 showed excellent tumor ablation in vivo. Therefore, this facile method provided an effective strategy for enhanced PDT treatment of ICG and showed great potential in clinical application. Electronic Supplementary Material: Supplementary material (measurements of the singlet oxygen quantum yield of ICG-NBs-O2, time-dependent temperature changes during the laser irradiation, photographs of Cal27 tumor-bearing nude mice and complete blood count of health male balb/c mice analysis) is available in the online version of this article at 10.1007/s12274-022-4085-0.

Stability of Oxygen Nanobubbles under Freshwater Conditions.

The use of nanobubbles (NBs) has gained significant attention in various applications (e.g., aeration in biological water treatment, water disinfection, membrane defouling, and ground water and sediment remediation) in recent decades because of their superior characteristics such as the improved mass transfer at the gas-liquid interfaces, their lifetime up to a couple of weeks, the formation of reactive oxygen species (ROS) with high oxidative potential. However, there is a lack of information about the effect of various factors on the stability of NBs for a long storage period under freshwater conditions. In this study, a comprehensive investigation was conducted to systematically examine the stability of oxygen NBs in water under various conditions which are closely related to a typical freshwater or the drinking water treatment. The oxygen NB stability in water was evaluated by monitoring the change in the bubble concentrations, size distribution, average diameter, and zeta potential for 60 days of storage time under different pH, hardness, ionic strength, natural organic matter (NOM), chlorine, and temperature conditions. In addition, the formation of hydroxyl radical (•OH) was investigated using disodium terephthalate which form fluorescent adducts with •OH in the presence of oxygen NBs. Among the parameters investigated, the impacts of cations, low pH, and high SUVA254 NOM on the stability of oxygen NBs were more significant than other conditions. The half-lives of oxygen NBs under various conditions follow the order Ca2+ < Na+ < pH 3 < high SUVA254 NOM < pH 5 < 30 °C. Oxygen NBs were more stable in softwater than hardwater. Oxygen NBs were relatively stable for 3 days regardless of pH. For a longer storage period, oxygen NBs disappeared faster at pH 3 than at high pH. High SUVA254 NOM destabilized NBs more than low SUVA254 NOM, indicating the impact of hydrophobicity on the NB stability. The temperature effect on the NB stability was negligible for a short storage time, while higher temperature destabilized oxygen NBs for a longer storage time. One of the main disappearance pathway of oxygen NBs in water was found to be coalescing, rising, and leaving the container, which would be promoted greatly by cations, low pH and NOM with high aromaticity. The formation of hydroxyl radical in NB solutions was detected at pH 3 by a florescent probe molecule. When oxygen NBs are released in water bodies, high calcium, high SUVA254 NOM, and low pH would significantly reduce the availability of NBs and their residence time in freshwater.

Graphene/CuO2 Nanoshuttles with Controllable Release of Oxygen Nanobubbles Promoting Interruption of Bacterial Respiration.

An oxygen nanoshuttle based on a reduced graphene oxide/copper peroxide (rGO/CuO2) nanocomposite has been presented to deliver in situ oxygen nanobubbles (O2 NBs) for combating bacterial infections. In the presence of rGO, the solid source of oxygen (i.e., CuO2) was decomposed (in response to environmental conditions such as pH and temperature) into O2 NBs in a more controllable and long-lasting trend (from 60 to 144 h). In a neutral buffer, the O2 NBs experienced growth and collapse evolutions, creating a dynamic micro-nanoenvironment around the nanocomposite. In addition to effective battling against methicillin-resistant Staphylococcus aureus bacteria, the O2 NBs demonstrated superior antibacterial properties on Gram-positive S. aureus to those on Gram-negative Escherichia coli bacteria, especially in the presence of rGO. In fact, the rGO contents could provide synergistic effects through harvesting some respiratory electrons (leading to striking interruption of the bacterial respiratory pathway) in one side and transferring them into the O2 NBs, resulting in nanoscale reactive oxygen species (ROS) generation in another side. Moreover, near-infrared laser irradiation induced more damage to the cell membrane due to the synergistic effects of local heat elevation and catalyzing the release/collapse of NBs imposing mechanical disruptions. Our results show that the O2-containing nanoshuttles can effectively be used as intelligent and controllable anti-infection nanorobots in upcoming graphene-based nanobiomedical applications.

Effective delivery of mycophenolic acid by oxygen nanobubbles for modulating immunosuppression.

Immunosuppressive drugs are crucial for preventing acute graft rejection or autoimmune diseases. They are generally small molecules that require suitable drug carriers for ensuring stability, bioavailability, and longer half-life. Mycophenolic acid (MPA) is an extensively studied immunosuppressive drug. However, it requires suitable carriers for overcoming clinical limitations. Currently, lipid-shelled micro- and nanobubbles are being thoroughly investigated for diagnostic and therapeutic applications, as they possess essential properties, such as injectability, smaller size, gaseous core, high surface area, higher drug payload, and enhanced cellular penetration. Phospholipids are biocompatible and biodegradable molecules, and can be functionalized according to specific requirements. Methods: In this study, we synthesized oxygen nanobubbles (ONBs) and loaded the hydrophobic MPA within the ONBs to generate ONB/MPA. Peripheral blood mononuclear cells (PBMCs) were treated with ONB/MPA to determine the suppression of immune response by measuring cytokine release. In vivo murine experiments were performed to evaluate the effectiveness of ONB/MPA in the presence of inflammatory stimulants. Results: Our results suggest that ONBs successfully delivered MPA and reduced the release of cytokines, thereby controlling inflammation and significantly increasing the survival rate of animals. Conclusion: This method can be potentially used for implantation and for treating autoimmune diseases, wherein immunosuppression is desired.

Anoxia remediation and internal loading modulation in eutrophic lakes using geoengineering method based on oxygen nanobubbles.

Benthic anoxia and internal P release, widely occurring in eutrophic lakes, are major factors threatening the health of aquatic ecosystems. In this paper, we experimentally evaluated the efficacy of a new type of "flock-lock" geoengineering method based on oxygen nanobubble technology to remediate sediment anoxia and reduce the internal P release in waters with and without algal blooms. Oxygen-carrying materials (OCM) modified from natural zeolites were used as capping agents and an oxygen-locking layer consists of OCM and the oxidized sediment was formed between anoxic sediment and overlying water. The synergy of diffusion and retention of oxygen in this layer contributes to both the increase of DO and reversal of anoxic conditions. By capping with OCM, the DO in overlying water improved instantly from around 1.5 mg/L to 3.5-4 mg/L and 5-6 mg/L in the systems with algal blooms and without algal blooms, respectively, and maintained throughout the incubation period. The oxygen penetration depth in the sediment can be significantly enhanced from around 0 cm to 3 cm and form an oxygen-locking layer at the end of the experiment by capping with OCM. The labile P was effectively retained via the re-oxidation of ferrous iron in this layer compared with the obvious release of labile P and Fe in control. More importantly, the oxygen depletion and labile P increase at the sediment-water interface caused by the decomposition of the deposited algal biomass can be substantially eliminated after capping with OCM. The study shed insights on the sustainable modulation of sediment anoxia and internal loading in eutrophic waters.

Anti-Tumor Drug-Loaded Oxygen Nanobubbles for the Degradation of HIF-1α and the Upregulation of Reactive Oxygen Species in Tumor Cells.

Hypoxia is a key concern during the treatment of tumors, and hypoxia-inducible factor 1 alpha (HIF-1α) has been associated with increased tumor resistance to therapeutic modalities. In this study, doxorubicin-loaded oxygen nanobubbles (Dox/ONBs) were synthesized, and the effectiveness of drug delivery to MDA-MB-231 breast cancer and HeLa cells was evaluated. Dox/ONBs were characterized using optical and fluorescence microscopy, and size measurements were performed through nanoparticle tracking analysis (NTA). The working mechanism of Dox was evaluated using reactive oxygen species (ROS) assays, and cellular penetration was assessed with confocal microscopy. Hypoxic conditions were established to assess the effect of Dox/ONBs under hypoxic conditions compared with normoxic conditions. Our results indicate that Dox/ONBs are effective for drug delivery, enhancing oxygen levels, and ROS generation in tumor-derived cell lines.

Surface Composition and Preparation Method for Oxygen Nanobubbles for Drug Delivery and Ultrasound Imaging Applications.

Phospholipids have been widely investigated for the preparation of liposomes, and micro and nanobubbles. They comprise biocompatible and biodegradable molecules and offer simple preparation with a variety of functions in diagnostic and therapeutic applications. Phospholipids require emulsifiers and surfactants to assemble in the form of bubbles. These surfactants determine the size, zeta potential, and other characteristics of particles. Polyethylene glycol (PEG) and its various derivatives have been employed by researchers to synthesize micro and nanobubbles. The stability of phospholipid-shelled nanobubbles has been reported by various researchers owing to the reduction of surface tension by surfactants in the shell. Nanobubbles have been employed to deliver oxygen to tissues and hypoxic cells. In this study, we investigated the effects of different ratios of phospholipids to PEG on the size, distribution, and characterization of oxygen nanobubbles (ONBs). ONBs were synthesized using a sonication technique. We analyzed and compared the sizes, numbers of generated particles, and zeta potentials of different compositions of ONBs using dynamic light scattering and nanoparticle tracking analysis. Then, we employed these oxygen nanobubbles to enhance the cellular microenvironment and cell viability. ONBs were also investigated for ultrasound imaging.

Lipid-Polymer Bilaminar Oxygen Nanobubbles for Enhanced Photodynamic Therapy of Cancer.

Hypoxia in solid tumors may be a hindrance to effective treatments of tumors in achieving their therapeutic potential, especially for photodynamic therapy (PDT) which requires oxygen as the supplement substrate. Oxygen delivery using perfluorocarbon emulsions or lipid oxygen microbubbles has been developed as the agents to supply endogenous oxygen to fuel singlet oxygen generation in PDT. However, such methods suffer from premature oxygen release and storage issues. To address these limitations, we designed lipid-polymer bilaminar oxygen nanobubbles with chlorin e6 (Ce6) conjugated to the polymer shell as a novel oxygen self-supplement agent for PDT. The resultant nanobubbles possessed excellent stability to reduce the risk of premature oxygen release and were stored as freeze-dried powders to avoid shelf storage issues. In vitro and in vivo experimental results demonstrated that the nanobubbles exhibited much higher cellular uptake rates and tumor targeting efficiency compared to free Ce6. Using the oxygen nanobubbles for PDT, a significant enhancement of therapeutic efficacy and survival rates was achieved on a C6 glioma-bearing mice model with no noticeable side effects, owing to the greatly enhanced singlet oxygen generation powered by oxygen encapsulated nanobubbles.

Engineering oxygen nanobubbles for the effective reversal of hypoxia.

Hypoxia, which results from an inadequate supply of oxygen, is a major cause of concern in cancer therapy as it is associated with a reduction in the effectiveness of chemotherapy and radiotherapy in cancer treatment. Overexpression and stabilization of hypoxia-inducible factor 1α (HIF-1α) protein in tumours, due to hypoxia, results in poor prognosis and increased patient mortality. To increase oxygen tension in hypoxic areas, micro- and nanobubbles have been investigated by various researchers. In the present research, lipid-shelled oxygen nanobubbles (ONBs) were synthesized through a sonication method to reverse hypoxic conditions created in a custom-made hypoxic chamber. Release of oxygen gas from ONBs in deoxygenated water was evaluated by measuring dissolved oxygen. Hypoxic conditions were evaluated by performing in vitro experiments on MDA-MB231 breast cancer cells through the expression of HIF-1α and the fluorescence of image-iT™ hypoxia reagent. The results indicated the degradation of HIF-1α after the introduction of ONBs. We propose that ONBs are successful in reversing hypoxia, downregulating HIF-1α, and improving cellular conditions, leading to further medical applications.

Oxygen Nanobubble Tracking by Light Scattering in Single Cells and Tissues.

Oxygen nanobubbles (ONBs) have significant potential in targeted imaging and treatment in cancer diagnosis and therapy. Precise localization and tracking of single ONBs is demonstrated based on hyperspectral dark-field microscope (HSDFM) to image and track single oxygen nanobubbles in single cells. ONBs were proposed as promising contrast-generating imaging agents due to their strong light scattering generated from nonuniformity of refractive index at the interface. With this powerful platform, we have revealed the trajectories and quantities of ONBs in cells, and demonstrated the relation between the size and diffusion coefficient. We have also evaluated the presence of ONBs in the nucleus with respect to an increase in incubation time and have quantified the uptake in single cells in ex vivo tumor tissues. Our results demonstrate that HSDFM can be a versatile platform to detect and measure cellulosic nanoparticles at the single-cell level and to assess the dynamics and trajectories of this delivery system.