Introduction

Myocardial infarction (MI), commonly known as a heart attack, remains one of the leading threats to global health. Currently, timely restoration of blood flow to ischemic myocardium is a critical treatment strategy for acute MI. However, this approach itself can trigger a pathological phenomenon known as myocardial ischemia/reperfusion injury (MI/RI). At the core of MI/RI lie intertwined processes of oxidative stress and inflammatory cascades, creating an urgent need for targeted therapies that address these molecular drivers.

Now, a pioneering study led by Professor Li Chun and Associate Professor Wang Jingyu at Beijing University of Chinese Medicine has achieved a significant breakthrough in integrating traditional Chinese and Western nanomedicine. Their study, titled "Artificial Cell Derived Vesicles from Ginsenoside Rg1-Primed Mesenchymal Stromal Cells Mitigate Oxidative Stress and DNA Damage in Myocardial Ischemia/Reperfusion Injury", has been published in the internationally renowned journal Nano Research (CAS Zone 1, Impact Factor 9.0). Addressing the critical challenge of MI/RI in acute MI treatment, the study innovatively combines the active herbal compound Ginsenoside Rg1 with modern nanobiotechnology to develop a novel nanoformulation: Rg1-ACDVs (Ginsenoside Rg1-induced artificial cell-derived vesicles), demonstrating high efficacy in mitigating myocardial ischemia/reperfusion injury.

This innovative approach not only represents a significant step forward in the precise treatment of heart attack complications but also serves as a compelling model for modernising traditional herbal medicine through interdisciplinary science. The Rg1-ACDV platform harnesses centuries-old medicinal knowledge and enhances it with contemporary biomedical engineering, opening new pathways in regenerative and nanomedicine. The research has been hailed as a paradigm-shifting contribution to both cardiology and integrative medicine, highlighting the potential of cross-disciplinary collaboration to solve complex clinical challenges.

Figure 1: Research Paper Published on Nano Research, titled “Artificial Cell Derived Vesicles from Ginsenoside Rg1-Primed Mesenchymal Stromal Cells Mitigate Oxidative Stress and DNA Damage in Myocardial Ischemia/Reperfusion Injury”

Research Background

Myocardial ischemia/reperfusion injury (MI/RI) remains a critical challenge in the treatment of acute myocardial infarction. Oxidative stress and inflammatory cascade reactions drive its underlying mechanisms, ultimately exacerbating cardiomyocyte death and contributing to heart failure. Although mesenchymal stromal cells (MSCs) and their derivatives offer promising immunomodulatory, anti-inflammatory, and tissue-repairing properties, challenges such as poor in vivo survival, insufficient targeted delivery efficiency, and difficulties in scalable production limit their clinical application. This study draws inspiration from traditional Chinese medicine, specifically Panax ginseng, long revered as the "king of herbs." Ancient texts such as the Compendium of Materia Medica (Bencao Gangmu) record its ability to "restore vital energy, nourish blood, strengthen the heart, and calm the mind," highlighting its traditional use in treating cardiovascular symptoms such as palpitations, exhaustion, insomnia, and chest pain. To enhance MSC function and therapeutic potential, the research team employed 3D dynamic cell culture using 3D TableTrix™ microcarriers and the 3D FloTrix™ miniSPIN FLEX 4-channel bioreactor from CytoNiche Biotech. This advanced culture system closely mimics the in vivo microenvironment, better preserving MSC stemness and biological functionality. Building on this platform, the team utilised ginsenoside Rg1, a key bioactive compound extracted from ginseng, to prime the 3D-cultured MSCs. The study revealed that Rg1 pretreatment significantly enhanced the proliferation, antioxidant capacity, and DNA repair function of 3D-cultured stem cells, laying a solid foundation for subsequent nanovesicles development. Finally, leveraging these Rg1-primed 3D-cultured MSCs, the team produced highly potent artificial cell-derived vesicles (Rg1-ACDVs) using a controlled process combining microporous continuous extrusion and ultracentrifugation. These bioengineered nanovesicles offer a promising, scalable, and robust cell

Study Design

Figure 2: Schematic Diagram of Rg1-ACDVs Preparation and their Cardioprotective Mechanism

The research design focused on optimising the function of Mesenchymal Stromal Cells (MSCs) in myocardial ischemia/reperfusion injury. Researchers achieved this goal by activating MSCs cultured on microcarriers within a 3D bioreactor system with ginsenoside Rg1. Membrane extrusion technology was then used to generate artificial cell-derived vesicles (ACDVs) from the Rg1-stimulated MSCs. The resulting Rg1-ACDVs demonstrated an enhanced therapeutic effect against myocardial ischemia/reperfusion injury by effectively reducing both oxidative stress and DNA damage. This novel approach successfully underscores the synergistic potential of traditional Chinese medicine and advanced nano-engineering for developing scalable cardiac therapies.

Data Analysis and Results

Figure 3: Enhanced Proliferation and Functional Characterisation of Ginsenoside Rg1-Primed Mesenchymal Stromal Cells in 2D and 3D Culture Systems

(a) IncuCyte imaging results demonstrate the change in MSC density in 2D culture plates following Rg1 treatment.

(b) Schematic diagram of MSC expansion on microcarriers within a 3D spinning bioreactor.

(c) Growth status of MSCs in 3D culture visualised by Calcein-AM and PI staining. 

(d) Quantification of MSC number over time in the 3D bioreactor. 

(e) Flow cytometry analysis confirms that MSC identity biomarkers remain unaffected by the culture process. 

(f) RNA sequencing reveals that Rg1 treatment exerts significant transcriptional effects on MSCs. 

(g) KEGG pathway analysis indicates that upregulated differentially expressed genes are enriched in cell cycle progression and DNA replication pathways.

(h) GSEA confirms a positive correlation between Rg1 treatment and gene sets associated with DNA repair and cell cycle regulation. 

Figure 4: Comprehensive Physical and Molecular Characterisation of Artificial Cell-Derived Vesicles (ACDV) and Extracellular Vesicles (EV) with and without Ginsenoside Rg1 Preconditioning

(a) Schematic illustration of ACDV production via extrusion and gradient centrifugation. 

(b) TEM images showing the morphology of ACDV, Rg1-ACDV, EV, and Rg1-EV. 

(c) NTA profiles showing the diameter distribution of the four types of vesicles. 

(d) NTA measurements of zeta potential.

(e) Western blot analysis of EV marker expression in ACDV, Rg1-ACDV, EV, Rg1-EV, and MSCs.

(f–g) miRNA-Seq analysis of ACDV, Rg1-ACDV, and Rg1-EV revealed: (f) Venn diagram showing differentially expressed miRNAs between groups;

(h) GO term enrichment analysis; and (g) GSEA results of genes targeted by DE-miRNAs between Rg1-ACDV and Rg1-EV. 

(i–k) Proteomic analysis of ACDV, Rg1-ACDV, EV, and Rg1-EV demonstrated: (i) Coomassie brilliant blue staining showing distinct protein expression patterns in ACDV and EV; (j) subcellular localisation analysis; and (k) KEGG pathway enrichment analysis of genes corresponding to differentially expressed proteins between Rg1-ACDV and Rg1-EV. 

Figure 5: Superior Protective Effects of Rg1-Primed Artificial Cell-Derived Vesicles (Rg1-ACDVs) Against H₂O₂-Induced Cardiomyocyte Injury In Vitro

(a–b) Cellular uptake of ACDVs by AC16 and H9c2 cardiomyocytes. 

(c–d) Relative cell viability of (c) AC16 and (d) H9c2 cells measured by CCK‑8 assay after 24‑h injury with gradient concentrations of H₂O₂. 

(e–f) Viability of (e) AC16 and (f) H9c2 cells after 24 hr H₂O₂ injury followed by various treatments, as assessed by CCK‑8. 

(g–j) Cell growth of AC16 (g–h) and H9c2 

(i–j) monitored by IncuCyte imaging over 24 h after H₂O₂ injury and different treatments. 

Figure 6: Therapeutic Efficacy of Rg1-Primed Artificial Cell-Derived Vesicles (Rg1-ACDVs) in a Rat Model of Myocardial Ischemia/Reperfusion Injury (MI/RI)

(a) Schematic of intramyocardial injection of Rg1-ACDVs for the treatment of MI/RI. 

(b) Cardiac function assessment by echocardiography, followed by analysis of (c) LVEF and (d) LVFS in rats. 

(e–f) Plasma levels of myocardial injury markers: (e) LDH and (f) CK-MB. 

(g–h) H&E staining of myocardial tissue. 

(i–j) Masson’s trichrome staining. 

(k) TUNEL staining showing apoptotic cells in myocardial tissue sections. 

Figure 7: Transcriptomic Analysis of AC16 Cells Reveals Rg1-ACDVs Mitigate DNA Damage and Oxidative Stress, Correlated with Enhanced Mitochondrial Transfer

(a–g) mRNA-Seq analysis of AC16 cells: (a) Volcano plot and (b) heatmap of differentially expressed genes (DEGs) identified between the model group and the control group. Comparative mRNA-Seq analysis of AC16 cells treated with Rg1-ACDVs versus Rg1-EVs: (c) Volcano plot, (d) GO term enrichment, (e) KEGG pathway enrichment, and (f) GSEA highlighting pathways associated with improved cell cycle progression and reduced DNA damage. 

(g) FPKM values of representative ROS marker genes. 

(h–i) Mito‑Tracker fluorescence imaging of H9c2 and AC16 cells, showing that Rg1‑ACDVs contain more mitochondrial components.

(j–k) Flow cytometric analysis of Mito‑Tracker fluorescence intensity in H9c2 and AC16 cells. 

Figure 8: Rg1-Primed Artificial Cell-Derived Vesicles (Rg1-ACDVs) Effectively Mitigate Global and Mitochondrial Oxidative Stress In Vitro and In Vivo

(a–b) Flow cytometry analysis of ROS levels in (a) AC16 and (b) H9c2 cells.

(c–d) Statistical analysis of MitoTracker and MitoSOX staining in AC16 and H9c2 cells, indicating reduced mitochondrial ROS following Rg1-ACDVs treatment. 

(e) Representative fluorescence images of DHE staining in rat myocardial tissue, demonstrating decreased ROS levels after Rg1-ACDVs treatment. 

(f–i) Plasma levels of oxidative stress markers: (f) SOD, (g) CAT, (h) GSH, and (i) MDA, showing enhanced antioxidant enzyme activity and reduced lipid peroxidation. 

Figure 9: Rg1-Primed Artificial Cell-Derived Vesicles (Rg1-ACDVs) Reduce DNA Damage and Oxidative Stress Markers (γ‑H2AX and 8-OHdG) in Cardiomyocytes In Vitro and Myocardial Tissue In Vivo 

(a–c) Representative immunofluorescence images showing γ‑H2AX and 8‑OHdG expression in H9c2 cells. 

(d–f) Representative immunofluorescence images showing γ‑H2AX and 8‑OHdG expression in AC16 cells. 

(g) Representative immunohistochemical image showing γ‑H2AX expression in myocardial tissue of SD rats.

Conclusion

This study demonstrates that preconditioning Mesenchymal Stromal Cells (MSCs) with ginsenoside Rg1 before producing Artificial Cell-Derived Vesicles (ACDVs) results in substantial therapeutic and manufacturing improvements compared to vesicles derived from conventionally cultured cells. 

The Rg1-ACDVs achieved significant improvements across several key dimensions: 

1. Higher Yield: Vesicle production increased nearly 10-fold, a critical metric enabling cost-effective, scalable manufacturing necessary for clinical translation. 
2. Enhanced Bioactivity: Enriched with proteins and miRNAs involved in DNA damage repair and oxidative stress regulation. 
3. Superior Therapeutic Effects: Functional analyses confirmed that Rg1-ACDVs significantly reduced myocardial injury, improved cardiac function, and decreased infarct size in both in vitro cellular and in vivo animal models.

Through integrated omics and functional analyses, this study systematically elucidated for the first time the mechanisms by which an active herbal compound enhances the therapeutic efficacy of nanovesicles, further revealing the core mechanistic advantages of Rg1‑ACDVs:

1. Multi‑Omics Empowerment: Rg1 upregulated multiple genes involved in DNA repair, cell cycle progression, and antioxidant pathways in stem cells. 
2. Upgraded Cargo Profile: Compared with untreated vesicles, Rg1‑ACDVs exhibited significantly higher levels of mitochondrial proteins and nuclear transcription‑regulatory proteins. 
3. Cardioprotective Efficacy: Rg1‑ACDVs markedly reduced reactive oxygen species (ROS) in cardiomyocytes and decreased expression of DNA damage markers γ‑H2AX and 8‑OHdG, demonstrating potent antioxidant and myocardial protective effects.

This work not only reveals that herbal monomers can act as “cellular activators” in the functional engineering of biological agents, but also establishes a modernised pathway for traditional Chinese medicine: “Herb → Active Compound → Cell Regulation → Functional Enhancement.”

Future Perspectives

Moving forward, the focus is on advancing the clinical applicability of these herb-activated nanovesicles through targeted delivery strategies. This critical next phase includes exploring formulations such as cardiac stent coatings and inhalable formulations, designed to provide more precise and effective therapeutic options for major cardiovascular diseases such as Myocardial Ischemia/Reperfusion Injury (MI/RI). This achievement is a clear exemplar of the research paradigm: “activating traditional wisdom with modern technology” through an integrated approach to Chinese and Western medicine. By starting with classical herbal knowledge and leveraging contemporary biotechnology to enhance the functional properties of therapeutic agents, this work offers a replicable and scalable model for Traditional Chinese Medicine's (TCM) participation in cutting-edge medical innovation. This integration not only validates the historical efficacy of traditional medicine but also underscores its significant disciplinary value and relevance in the evolving era of regenerative medicine. 

Author Information

Corresponding Author: Li Chun, Professor and Ph.D. Supervisor, Beijing University of Chinese Medicine. Co-corresponding Author: Wang Jingyu, Associate Professor and Master’s Supervisor, Beijing University of Chinese Medicine 

Research Technology Support

The advanced 3D cell manufacturing technologies developed by CytoNiche Biotech fundamentally enabled the groundbreaking study on Rg1-ACDVs. These systems provided the critical tools to realise the enhanced therapeutic potential of Mesenchymal Stromal Cells (MSCs), which underpin the novel Rg1-ACDV platform.

3D TableTrix™ Microcarriers

The research team employed 3D TableTrix™ microcarriers to create a physiologically relevant expansion environment for MSCs. The technology's unique features directly addressed key limitations in stem cell culture:  

1. Enhanced Biomimicry – Comprising tens of thousands of elastic, three-dimensional porous microcarriers with porosity >90%, controllable particle size ranging 50–500 μm, and uniformity ≤100 μm, providing a truly biomimetic 3D culture environment. 
2. Comprehensive Regulatory Approvals – Holds two CDE (Center for Drug Evaluation) drug excipient registrations (Registration Nos. F20200000496; F20210000003) and three FDA drug substance/excipient authorisations (Registration Nos. DMF: 037798, 035481; MF: 29721).
3. Efficient Harvesting – Proprietary degradable technology enables gentle, high-efficiency microcarrier cleavage, facilitating smoother cell recovery than conventional methods. 
4. Superior Safety Profile – Supported by authoritative quality and safety evaluation reports, including residual degradation product detection, cytotoxicity, pyrogenicity, genotoxicity, in vivo immunotoxicity, hemolysis, local dermal irritation, systemic anaphylaxis, and intraperitoneal injection toxicity. 
5. Scalability – Through 3D culture integrated with the full suite of CytoNiche's 3D cell manufacturing platform, fully automated, closed-system large-scale cell expansion can be achieved, yielding up to tens of billions of cells. 

3D FloTrix™ miniFLEX 4-Channel Bioreactor

The research team utilized the 3D FloTrix™ miniFLEX 4-channel bioreactor to perform the dynamic culture of MSCs, leveraging its precise process control to optimize cell growth and function:

1. Ultra-Slim Design – The main unit is only 48 mm tall, with a flat design that optimizes the utilisation of incubator space. 
2. Stainless-Steel Construction – Fully stainless-steel housing ensures easy cleaning and meets stringent cleanliness requirements. 
3. Compatible with Single-Use Vessels – The system is compatible with CytoNiche’s proprietary gas-permeable, built-in-impeller single-use bioreactor bottles. 
4. Researchers can place two bioreactor units – each hosting a total of 8 bottles in 125/250 ml vessels—within a standard 170 L incubator. 
5. Synchronisation Function – The linkage feature, combined with preset process protocols, enables multiple bottles to operate under a unified set of parameters. A single command synchronises start/pause/stop across all linked bottles, enabling efficient comparative experiments. 
6. Preset Process Profiles – Includes 10 built-in protocols and 4 customisable process modes, supporting multi-step and cyclic operations. Parameters are adjustable, retained after power loss, with single-step durations up to 1000 min and up to 100 repeat cycles. 
7. Programmable Agitation – Stirring programs are fully customisable, supporting intermittent, dual-speed, and intermittent-constant speed modes for process optimisation. Bilingual System Interface – Supports both Chinese and English language display with one-touch switching. 
8. Real-Time Monitoring – Continuously displays operational parameters per bottle: time, agitation speed, active process, and current step. 

References

Zhao, S., Fan, H., Yang, S., Xu, C., Liu, Y., Guo, Y., Yu, Y., Sun, Y., Li, H., Wang, Y., Guo, J., Li, C., & Wang, J. (2024). Artificial cell derived vesicles from Ginsenoside Rg1-primed mesenchymal stromal cells mitigate oxidative stress and DNA damage in myocardial ischemic/reperfusion injury. Nano Research, 18(6), 94907535. https://doi.org/10.26599/nr.2025.94907535