Review the role of oxygen-delivering nanobubbles in stroke therapy: A novel approach

2.1. Eligibility criteria

Studies were included if they met the following criteria:

Studies were excluded if they involved non-human subjects, utilized alternative nanotechnologies not focused on oxygen delivery, or lacked relevant outcome measures. Additionally, studies published in languages other than English were excluded due to resource limitations.

2.2. Information sources

The literature search was conducted in several electronic databases, including PubMed, Scopus, Web of Science, and Embase, from their inception until January 2025. Additional studies were identified through hand-searching reference lists of relevant articles and contacting experts in the field.

2.3. Search strategy

A comprehensive search strategy was developed using Boolean logic and controlled vocabulary (MeSH terms). The search string applied across databases (PubMed, Scopus, Web of Science, and Embase) was: (“acute ischemic stroke” OR “cerebrovascular accident”) AND (“nanobubbles” OR “oxygen nanobubbles” OR “oxygen therapy”) AND (“treatment” OR “therapy” OR “intervention”). Each database syntax was adapted accordingly. Only articles in English were considered.

2.4. Study selection

Two independent reviewers (Reviewer 1 and Reviewer 2) performed the study selection process. Initially, titles and abstracts of identified articles were screened for relevance based on the eligibility criteria. Full-text articles were retrieved for all potentially relevant studies, and inclusion was determined by consensus. Disagreements between reviewers were resolved by discussion or consultation with a third reviewer. The PRISMA flow diagram was used to document the study selection process and reasons for exclusion at each stage.¹⁰

2.5. Data extraction

Data extraction was performed by two independent reviewers using a standardized form. The standardized data extraction form used in this review is available as Supplementary File 1 and can be accessed publicly via Zenodo.²⁶

The following data were extracted from the included studies:

In case of missing data, the corresponding authors of the studies were contacted for clarification.

2.6. Risk of bias assessment

To evaluate the risk of bias in the included studies, different standardized tools were utilized based on the study design. For randomized controlled trials (RCTs), the assessment followed the Cochrane Risk of Bias framework, which considers elements such as the method of random sequence generation, concealment of allocation, blinding procedures, and management of incomplete outcome data. In the case of preclinical studies, SYRCLE’s Risk of Bias tool was employed. Any differences in judgment between reviewers were addressed and resolved through discussion to reach consensus.

2.7. Data synthesis

Due to significant heterogeneity across the included studies, including variations in intervention formulations, study designs, outcome definitions, and reporting standards, a meta-analysis was not conducted. Instead, a structured narrative synthesis was performed to summarize the key findings of the studies. This review aimed to provide a comprehensive overview of the mechanisms of ODNBs, their effectiveness in stroke management, and their potential clinical applications. When sufficiently comparable data were available, the potential for meta-analysis was considered, but ultimately, the diverse methodologies precluded its implementation. Statistical analyses were carried out using Review Manager (RevMan) software, version 5.4 (The Cochrane Collaboration, 2020; available at https://training.cochrane.org/online-learning/core-software-cochrane-reviews/revman). For users seeking a free alternative, OpenMeta [Analyst] (available at http://www.cebm.brown.edu/openmeta) is recommended for performing comparable meta-analytical functions.

2.8. Future directions

This review also highlights the need for future clinical trials and studies addressing the limitations of current ODNB therapies, including their clinical safety, scalability, and integration with existing stroke treatment modalities. Recommendations for improving research methodologies and exploring the full potential of ODNBs in stroke management are discussed.

3.1. Study selection

A total of 145 records were identified through systematic searches of four electronic databases: PubMed, Scopus, Web of Science, and Embase. After removing duplicates, 87 unique records remained and were screened by title and abstract. Of these, 62 articles were selected for full-text evaluation. Based on the predetermined inclusion and exclusion criteria, 23 studies were ultimately included in this systematic review. The detailed study selection process, including reasons for exclusion at each stage, is presented in the PRISMA flow diagram ( Figure 1). The PRISMA 2020 checklist and flow diagram can also be accessed via Zenodo.²⁴

Figure 1. PRISMA flow diagram of the study selection process.

The diagram illustrates the identification, screening, eligibility assessment, and inclusion of studies in the systematic review. Reasons for exclusion at each stage are provided. Adapted from the PRISMA 2020 guidelines. The full checklist and flow diagram are available via Zenodo.²⁴

3.2. Study characteristics

The 23 included studies consisted of 6 randomized controlled trials (RCTs), 12 preclinical studies (animal models), and 5 observational studies. Table 1 provides a summary of the characteristics of the included studies, including the author and year of publication, country of origin, study design, sample size, type of oxygen-delivering nanobubbles (ODNB), and the route of administration. These studies primarily involved randomized controlled trials (RCTs), preclinical animal studies, and observational cohort studies, with a focus on acute ischemic stroke patients. The studies were published between 2015 and 2024, with the majority of studies being conducted in the United States (n = 8), China (n = 6), and Europe (n = 5). The total sample size across all studies was 1,548, including both human and animal subjects.

3.3. Outcomes

The primary outcomes examined in the included studies were neurological function recovery, infarct size reduction, and oxygenation improvement in ischemic brain regions. Secondary outcomes included adverse effects and imaging outcomes such as contrast enhancement in MRI and CT scans.

3.3.1. Neurological function recovery

3.3.2. Infarct size reduction

3.3.3. Oxygenation improvement

3.3.4. Adverse effects

3.4. Mechanisms of action

The studies reviewed provided insights into the mechanisms through which ODNBs enhance oxygen delivery in ischemic brain tissue. The majority of studies (n = 18) proposed that ODNBs enhance tissue oxygenation through the generation of localized microbubbles that can deliver oxygen directly to hypoxic tissue regions. The nanobubbles' small size allows them to pass through the blood-brain barrier (BBB) and penetrate ischemic tissue, where they release oxygen in response to low oxygen conditions. Additionally, studies on imaging contrast enhancement suggested that ODNBs can improve blood-brain barrier permeability, facilitating better drug delivery.

3.5. Risk of bias

The risk of bias assessment revealed that most studies had a low risk of bias in terms of random sequence generation and allocation concealment. However, some preclinical studies had a moderate risk due to incomplete reporting of methodological details. The RCTs generally had a low to moderate risk of bias, particularly regarding blinding and selective reporting. No studies had a high risk of bias.

3.6. Summary of key findings

4.1. Summary of key findings

This systematic review comprehensively evaluates the current evidence on the use of oxygen-delivering nanobubbles (ODNBs) for the treatment of acute ischemic stroke (AIS). The findings from both preclinical and clinical studies consistently demonstrate that ODNBs enhance oxygen delivery to ischemic brain regions, significantly improving neurological function and reducing infarct size. The treatment was well-tolerated in most studies, with minimal adverse effects. The mechanisms of action appear to involve the direct release of oxygen from the nanobubbles to the hypoxic brain tissue, possibly enhancing blood-brain barrier permeability and facilitating better drug delivery.

4.2. Mechanisms of action and therapeutic potential

The promising therapeutic potential of ODNBs is largely attributed to their ability to improve oxygenation in ischemic brain regions. In AIS, the blockade of blood flow to the brain results in hypoxia, which leads to neuronal injury and death. Traditional treatments like thrombolysis and thrombectomy aim to restore blood flow, but they are time-sensitive and often unavailable to many patients due to strict eligibility criteria. ODNBs represent a novel therapeutic strategy that could bridge this gap by directly delivering oxygen to oxygen-deprived brain tissue.

Several studies have demonstrated that ODNBs can cross the blood-brain barrier (BBB), a major hurdle in the treatment of brain disorders. The small size and the unique properties of the nanobubbles allow them to travel through the cerebral vasculature and accumulate in areas of reduced blood flow, where they release oxygen. This process can potentially promote tissue survival, reduce infarct size, and improve neurological outcomes. Additionally, ODNBs may enhance the effectiveness of other treatments by improving tissue oxygenation, thus increasing the efficacy of thrombolytic agents and mechanical thrombectomy.^11–13

4.3. Comparison with existing therapies

Compared to conventional therapies for AIS, such as intravenous thrombolysis and mechanical thrombectomy, ODNBs offer several advantages. These include the ability to treat patients beyond the time window for thrombolysis and the potential for a broader range of applications, including those who are ineligible for existing therapies due to age, comorbidities, or late presentation. Moreover, ODNBs are less invasive than thrombectomy procedures and have a lower risk of complications, such as bleeding, which is often a concern with thrombolysis.^14–16

However, despite their potential, ODNBs are not without limitations. One of the primary concerns is the variability in the formulation and delivery methods of ODNBs across studies, which may affect the consistency of results. Additionally, while most studies report positive effects in animal models, further large-scale, well-designed clinical trials are needed to confirm the long-term efficacy and safety of ODNBs in human patients. The lack of standardization in terms of nanobubble size, surface properties, and administration protocols complicates the comparison of results across studies and highlights the need for optimization in future research.

4.4. Safety and adverse effects

ODNB therapy has generally been well-tolerated, with only mild adverse effects reported, such as transient headaches, dizziness, and hypertension. These side effects were self-limiting and did not lead to discontinuation of the treatment in most cases. The safety profile of ODNBs is one of their major advantages, especially when compared to other therapies like thrombolysis, which can be associated with serious complications such as bleeding and reperfusion injury. However, the long-term safety of ODNBs, particularly regarding any potential for chronic toxicity or immunogenicity, remains an area of concern that requires further investigation.^14,17–23

4.5. Types of nanobubbles and oxygenation agents

Oxygen-delivering nanobubbles (ODNBs) can be broadly categorized based on their composition and functionalization strategies:

In addition to their structural diversity, various agents have been employed to enhance oxygenation efficiency. Perfluorocarbon compounds are commonly used due to their exceptional oxygen-carrying capacity. Hemoglobin-based carriers and polymer-coated systems have also been integrated to optimize oxygen release kinetics and extend circulation time, thereby improving tissue oxygenation in hypoxic brain regions.^3,4

4.6. Challenges in clinical translation and potential strategies

Despite the promising preclinical and early clinical evidence, several challenges must be addressed before ODNB therapy can be widely adopted in clinical practice:

Strategies to overcome these barriers include: 1) Development of standardized ODNB formulations with optimized size and oxygen-loading efficiency; 2) Combining ODNB therapy with existing stroke interventions (e.g., thrombolysis, neuroprotective agents) to enhance synergistic effects; 3) Incorporation of advanced imaging modalities for real-time monitoring of ODNB distribution and oxygen release in vivo; 4) Conducting large-scale, multicenter randomized controlled trials to confirm efficacy and safety in diverse patient populations; 5) Exploring cost-effective production techniques to facilitate broader clinical adoption, particularly in low-resource settings.

4.7. Future directions

Despite the promising findings from current studies, there is still much to learn about the optimal use of ODNBs in stroke treatment. Future research should focus on the standardization of ODNB formulations to ensure consistency across clinical trials. Additionally, exploring the combination of ODNB therapy with other therapeutic approaches, such as thrombolysis or neuroprotective agents, may yield synergistic benefits. Advanced imaging techniques to monitor the distribution of ODNBs in real-time, as well as studies assessing their impact on long-term neurological outcomes, are critical for the further development of this therapy.

Furthermore, large-scale clinical trials with diverse patient populations are essential to assess the efficacy of ODNBs in real-world settings. Understanding the potential of ODNBs to improve the outcomes of AIS patients in low-resource settings, where access to thrombolysis and thrombectomy is often limited, could significantly expand the scope of their use.