Intelligent drugs based on notch protein remodeling: a defensive targeting strategy for tumor therapy

The delivery of intravenously administered therapeutic drugs to tumor sites is often impeded by biological barriers, including intravascular, endothelial, and extracellular matrix barriers. These barriers prevent efficient drug delivery and lead to drug accumulation in healthy tissues, causing significant side effects. Endostatin, for instance, while effective against various cancers, exhibits widespread tissue distribution, with 90-95% accumulating in healthy organs, causing potential cardiotoxicity. Traditional targeted therapies, like antibody-drug conjugates and cell carriers, aim to increase drug concentration at the tumor site but still struggle to overcome biological barriers and minimize off-target effects. Antibody-drug conjugates lack chemotactic abilities to reach tumor sites effectively, and cell carriers, while able to recognize tumor-released cytokines, may be susceptible to similar microenvironmental cues in healthy tissues. This research proposes a 'defensive targeting' strategy to mitigate off-target effects by creating an intelligent drug that autonomously distinguishes between tumor and healthy cells. This intelligent drug leverages the modular nature of the Notch signaling pathway, enabling customization of cell responses to specific tumor signals. The study uses Her2+ tumors and endostatin to develop and test this novel approach.

The authors reviewed existing literature on the challenges of drug delivery in cancer treatment, highlighting the limitations of traditional targeted therapy strategies. They cited studies demonstrating the efficacy of endostatin against various cancers, while also noting its significant cardiotoxicity due to widespread distribution. Previous research on antibody-drug conjugates and cell carriers was reviewed, underscoring their limitations in fully overcoming biological barriers. Existing literature on the Notch signaling pathway and its modularity, enabling customization of cell signaling, was also examined. This review provided the foundation for the development of an intelligent drug based on MSCs and the Notch signaling pathway.

The study involved the engineering of mesenchymal stem cells (MSCs) to create an intelligent drug called iMSCEndostatin. This involved modifying the Notch protein within MSCs by replacing its extracellular and intracellular domains with Her2-affibody (for Her2+ tumor recognition) and GAL4-VP64 (for regulating endostatin expression), respectively. A Gal4/UAS-endostatin response structure was incorporated to enable endostatin production upon tumor recognition. The modified MSCs (iMSCEndostatin) were generated using lentiviral-mediated transfection. In vitro studies involved co-culturing iMSCEndostatin with various cell lines, including Her2+ tumor cells (BT474, Skov3) and normal tissue cells (293T, BJ, L02, Svog, HUVEC, AC16), to assess its tumor recognition capabilities. Endostatin production and deployment were assessed using fluorescence microscopy, live-cell imaging, flow cytometry, and ELISA. The effect of iMSCEndostatin on angiogenesis was evaluated using CCK-8 assays, flow cytometry (CD105 expression), tube formation assays, and matrigel plug assays. In vivo studies used Emt-6 cells transfected with human Her2 and GFP to establish solid tumor and lung metastasis models in mice. iMSCEndostatin efficacy was evaluated by monitoring tumor growth, vascular density, and cardiotoxicity.

iMSCEndostatin demonstrated highly specific recognition of Her2+ tumor cells in vitro, distinguishing them from various normal cell types, including cardiac cells. Confocal microscopy and live-cell imaging confirmed that iMSCEndostatin specifically targeted and localized around Her2+ tumor cells, releasing endostatin only at the tumor site. Quantitative analysis revealed that iMSCEndostatin produced and released significant amounts of endostatin (approximately 0.62 pg per 5x10^4 cells in 24 h). In vitro experiments showed that activated iMSCEndostatin, triggered by tumor recognition, significantly inhibited angiogenesis, as evidenced by reduced HUVEC viability, CD105 expression, and tube formation. In vivo studies using solid tumor and lung metastasis models demonstrated that iMSCEndostatin effectively inhibited tumor growth and angiogenesis without inducing detectable cardiotoxicity. These findings confirmed that iMSCEndostatin successfully delivers endostatin specifically to the tumor site, significantly impacting tumor growth while minimizing side effects.

The study successfully demonstrated the feasibility of a defensive targeting strategy for drug delivery in cancer treatment. The intelligent drug iMSCEndostatin, by specifically targeting and releasing its payload only at the tumor site, overcomes the limitations of traditional targeted therapies. The modular design of iMSCEndostatin offers significant advantages, enabling customization to various tumor types and therapeutic agents. The use of MSCs as a delivery vehicle offers a unique advantage due to their homing capabilities and avoidance of tumor-induced immunosuppression. The lack of observed cardiotoxicity in vivo highlights the safety profile of this approach. The study's findings provide a promising foundation for the development of next-generation intelligent drugs for personalized cancer treatment.

This study introduces a novel approach to cancer therapy using intelligent drugs based on engineered MSCs. iMSCEndostatin, through its precise and targeted release of endostatin at the tumor site, effectively inhibits tumor growth and angiogenesis while minimizing off-target toxicity. The modular design of this intelligent drug platform holds great promise for personalized cancer therapies. Future research could focus on exploring its efficacy in different cancer types and evaluating the long-term effects and safety profiles. Investigating the integration of multiple response programs within iMSCEndostatin to address more complex therapeutic needs is also a promising avenue for future investigation.

The study primarily focused on Her2+ tumors, limiting its generalizability to other cancer types. The in vivo studies were conducted using a relatively small sample size. Long-term efficacy and safety studies are warranted to fully assess the potential clinical applications of iMSCEndostatin. Further research is also needed to optimize the production and delivery of iMSCEndostatin for clinical translation.