Worldwide, hypoxia-related conditions, including cancer, COVID-19, and neuro-degenerative diseases, often lead to multi-organ failure and significant mortality. Oxygen, crucial for cellular function, becomes scarce as levels drop below 10 mmHg (<2% O2), triggering mitochondrial dysregulation and activating hypoxia-induced factors (HiFs). Herein, oxygen nanobubbles (OnB), an emerging versatile oxygen delivery platform, offer a novel approach to address hypoxia-related pathologies. This review explores OnB oxygen delivery strategies and systems, including diffusion, ultrasound, photodynamic, and pH-responsive nanobubbles. It delves into the nanoscale mechanisms of OnB, elucidating their role in mitochondrial metabolism (TFAM, PGC1alpha), hypoxic responses (HiF-1alpha), and their interplay in chronic pathologies including cancer and neurodegenerative disorders, amongst others. By understanding these dynamics and underlying mechanisms, this article aims to contribute to our accruing knowledge of OnB and the developing potential in ameliorating hypoxia-and metabolic stress-related conditions and fostering innovative therapies.
Introduction

Hypoxia and Mitochondrial Dysfunction: Interplay and Implications in Pathology(ies)

Herein, the hypoxia-inducible factor or HiF is a heterodimeric transcription factor that plays a central role in cellular adaptation to low oxygen levels. It is considered a master regulator of the cellular response to hypoxia and plays a crucial role in maintaining oxygen homeostasis in various tissues and cells [29]. Briefly, the HiF is composed of two sub-units: HiF-α (alpha) and HiF-β (beta). The alpha subunit is oxygen-sensitive and exists in different isoforms, with HiF-1α and HiF-2α being the most well-known. In normoxic (oxygen-rich) conditions, prolyl hydroxylase enzymes modify HiF-α, marking it for degradation. However, in hypoxic conditions, this degradation is inhibited, allowing HiF-α to accumulate and form a heterodimer with HiF-β. This active HiF complex then translocates to the cell nucleus, where it binds to specific DNA sequences known as hypoxia-response elements or HREs and initiates the transcription of genes involved in various adaptive responses to low oxygen, including angiogenesis, erythropoiesis, glycolysis, and more. It is noteworthy to mention that the HiF ability to regulate the expression of genes involved in oxygen homeostasis and adaptation to hypoxia makes it a critical factor in various physiological and pathological processes, including embryogenesis, ischemic diseases, cancer, and metabolic disorders. Understanding the role of HiF and its downstream targets is essential for gaining insights into how cells and organisms respond to changing oxygen levels and for developing therapeutic strategies to address related diseases and conditions [29]. Remember that once the oxygen decreases below physiological levels, cells trigger a hypoxic cell response, characterized by the HiF-1α stabilization and its nuclear transcription (Figure 3A). Consequently, the translocation of HiF-1α leads to the activation of approximately 400 target genes. This activation influences metabolic processes by upregulating proteins associated with glycolysis while concurrently suppressing oxygen-dependent pathways, particularly those related to mitochondrial metabolism [30,31]. These findings establish a direct connection between mitochondria and HiF pathway, highlighting interdependence in response to changes in oxygen supply and demand.

Nanobubbles in Biomedicine: Bridging Basic Fundamentals to Practical Application(s)
3.1. Bubble Size and Physico-Chemico-Mechanical Properties
3.2. Structural Composition and Electrostatic Charge of OnB Affects Gas Core and Diffusion
NanoBubbles as a Platform for O2 Delivery: Innovative OnB-Mediated Oxygenation
Molecular Insights into the Mechanism of OnB in Chronic Diseases and Disorders
5.1. OnB and Neurodegenerative Disorders/Diseases
5.1.1. Role of OnB in Increasing/Enhancing Mitochondrial Bio-Genesis and Cellular Energy
5.1.2. Mitochondrial Bio-Genesis via the Phosphatidylinositol 3-Kinase (PI3K) Enzyme
5.2. OnB and Overcoming Hypoxia and Hypoxic Conditions
5.2.1. Molecular Bases to Downregulate/Suppress HiF-1α Post-OnB Application/Therapy
5.2.2. OnB and Epigenetic Modulation in Tumors
Other Remarks
Conclusions
The use of ultrafine bubbles as customizable nanocarriers or platforms infused with gas/drug-based nanotechnology represents a highly promising frontier, particularly in the context of conditions related to oxygen deficiency, such as neurodegenerative disorders, cardiovascular diseases, and cancer. Nanobubbles offer a range of advantages, including their stability for oxygenation, efficient gas exchange within confined volumes, and prolonged gas delivery compared to conventional micro/macro bubbles. These attributes encompass negative electric surface charge density, extended lifespan, reduced buoyancy, the ability to carry multiple gases, and strong echogenicity. Both coated and uncoated OnB have shown significant potential in enhancing mitochondrial metabolism, overcoming hypoxia, and increasing oxygen availability across various diseases, including cancer, neurodegenerative disorders, and chronic inflammation. Coated nanobubbles have garnered specific attention for their versatility in carrying not only gases but also various molecules, including drugs, proteins, DNA, and ligands, for precise in vivo delivery. While existing evidence mainly consists of in vitro studies highlighting the robustness of OnB in cellular oxygenation and their ability to downregulate HiF-1α while enhancing mitochondrial function through PI3K-mediated increases in PGC1α and TFAM transcription, several aspects require further exploration. These include the measurement of oxygen dynamics within cells, investigations into antioxidant cell signaling (such as SOD2), assessments of mitochondrial health and fitness, and comprehensive evaluations of biosafety and targeting precision in preclinical and clinical trials, especially for intravenous OnB applications. Additionally, uncharted territories such as muscular recovery, cutaneous lesion oxygenation, and tissue regeneration offer exciting prospects for future research. Although some lipid-shelled microbubbles (1–10 μm, OmB) are already in widespread clinical use as contrast agents for echocardiography, concerning the translational barriers associated with nanobubble-mediated oxygenation in cancer treatment and other chronic diseases, these methods appear to grapple some pivotal challenges. Firstly, there is an issue with pre-mature oxygen release and storage inadequacies, wherein only a limited amount of oxygen can be delivered, or gas generation would prove insufficient. Moreover, these approaches must address the second challenge, which pertains to ensuring size stability at the submicron level. This stability is critical for enhancing nanobubble accumulation in tumors, with size being the primary advantage of nanobubbles over their larger counterparts. Thirdly, achieving precise control over gas/drug release emerges as another critical aspect. This control is essential for tailoring responsive nanobubbles effectively to stimuli such as acoustic pressure, a well-explored mechanism that has opened new avenues in both the diagnosis and therapy of cancer and other diseases. Looking ahead, nanobubbles also present a range of other exogenously responsive mechanisms to environmental or external stimuli, such as light, temperature changes, electric and magnetic fields, and endogenous triggers like pH and redox states. These features align well with the versatile array of biomaterials (lipids, surfactants, polymers, and proteins), opening doors to diverse applications in theragnosis, drug design, and delivery. In summary, oxygenation by nanobubbles offers a promising strategy for addressing diseases characterized by hypoxia and mitochondrial dysfunction. It has the potential to improve oxygen delivery to specific tissues and enhance therapeutic outcomes while minimizing systemic side effects. However, addressing stability, biocompatibility, and regulatory concerns is critical for its successful translation into clinical practice. As research in this field advances, nanobubble-mediated oxygenation may emerge as a valuable tool in the treatment of various diseases.