(562i) Engineered Virus-like Vesicles for In Vivo Targeted Drug Delivery in TNBC Treatment

Triple-negative breast cancer (TNBC) is among the most challenging types of cancers to treat, due to the absence of receptor expression, including estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, which are common targets for hormone therapies. Chemotherapy remains the primary treatment for TNBC; yet, its efficacy is limited by drug resistance, severe side effects, and high toxicity levels. Exploring alternative therapeutic approaches, scientists are targeting the "glutamine addiction" and metabolic shift known as the Warburg Effect, where TNBC cells rely heavily on glutamine for energy. By inhibiting glutaminase (GLS), the enzyme essential for converting glutamine into glutamate, this strategy aims to starve TNBC cells of a critical resource for their growth. Despite the promise of GLS inhibitors, their poor water solubility leads to suboptimal potency, bioavailability, and circulation time, compromising their effectiveness in suppressing tumor growth. While various carriers have been developed to overcome drug delivery limitations, challenges such as high cytotoxicity, rapid clearance, and poor targeting capacity persist.

Addressing this challenge, our study utilized leukocyte-derived extracellular vesicles (EVs) known for their natural ability to target tumors. We aim to directly deliver hydrophobic GLS inhibitors to the tumor's inflammatory environment. Despite the biocompatibility and innate delivery capability of leukocyte EVs, effective encapsulation of drugs and vesicle uptake by recipient cells remain significant hurdles. Here we genetically modified donor cells to include virus-like capsid proteins inside the EVs to encapsulate drugs, along with envelope proteins on the EV surface to enhance cellular uptake by target cells. Additionally, we introduced an RNA stabilizer to form hydrophobic pockets within these capsids, significantly enhancing the ability of the EVs to enclose and maintain GLS inhibitors. These inhibitors were simply added to the culture of donor leukocytes, which then naturally incorporated them into the capsids within the EVs they produced. To evaluate the efficacy of engineered EVs in carrying GLS inhibitors, we used fluorescent inhibitors and vesicle lipid membrane dyes for easy tracking and visualization within recipient cells. More importantly, to assess its effectiveness against TNBC in vivo, we established a murine xenograft model by injecting human TNBC cells into immune deficient mice, closely replicating the human disease environment. We then administered the engineered EVs intravenously in a controlled dosage schedule to ensure maximum impact with minimal side effects, monitoring tumor growth and assessing the treatment's effectiveness through caliper measurements and final analysis of tumor sizes and weights after euthanizing the mice.

As a result, we demonstrated that engineered vesicles enriched within the tumor, successfully encapsulated, and transported hydrophobic GLS inhibitors into TNBC cells. Our in vivo studies demonstrated that systemic delivery of engineered EVs significantly reduced the size of TNBC tumors. Specifically, mice treated with EVs loaded with GLS inhibitors showed a 53% decrease in tumor volume and a notable extension in lifespan, highlighting the effectiveness of this novel delivery system in addressing TNBC. This new approach leverages the unique virus-like features of engineered nanocarriers, which mimic natural viral mechanisms of cellular entry and infection, to enhance therapeutic delivery and efficacy significantly. By integrating virus-like components into EVs, our research paves a new pathway for the encapsulation and targeted delivery of hydrophobic drugs into the inflammatory microenvironments of various pathological conditions, such as cancers and neurodegenerative diseases.