(W1230-07-41) Mesenchymal Stem Cell Derived Exosomes as Nanocarriers for Paclitaxel in Pancreatic Ductal Adenocarcinoma (PDAC) Treatment

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers, with a 5-year survival rate of only about 13%. This low rate is primarily due to late diagnosis, poor response to therapies, and a high degree of invasiveness. Despite advancements in surgery, chemotherapy, immunotherapy, and targeted therapies, conventional treatments often prove ineffective in advanced stages of the disease. This ineffectiveness can be attributed to inadequate tumor targeting, chemotoxicity, and drug resistance. Paclitaxel (PTX), a commonly used chemotherapeutic agent, works by stabilizing microtubules and halting cell division. However, its clinical efficacy in treating PDAC is often limited by poor solubility, low bioavailability, and severe dose-limiting side effects. Exosomes—small extracellular vesicles measuring 50 to 200 nanometers—have emerged as promising nanocarriers for drug delivery. This is largely due to their natural role in intercellular communication and their ability to carry bioactive molecules. Exosomes derived from mesenchymal stem cells (MSCs) offer additional benefits, including biocompatibility, immune evasion, and inherent tumor-homing capabilities. This study aims to investigate the potential of MSC-derived exosomes as a delivery system for PTX in PDAC treatment. We hypothesize that using exosomes to deliver PTX will enhance the drug's accumulation at tumor sites, reduce systemic toxicity, and improve therapeutic outcomes in pancreatic cancer.

Methods: MSCs were cultured under standard conditions, and exosomes were isolated from the conditioned media using a tangential flow filtration (TFF) system. The isolated exosomes were characterized by transmission electron microscopy (TEM) to assess their morphology, Zetasizer to measure their hydrodynamic diameter and surface charge, and nanoparticle tracking analysis (NTA) to confirm their size and concentration further. The identification of exosomes was confirmed by the surface expression of CD63 and CD81 using flow cytometry. Similarly, CD90 was used to verify their MSC origin. Protein quantification was performed using the bicinchoninic acid (BCA) assay. The biocompatibility of the exosomes was evaluated by culturing pancreatic cancer (PANC-1 and BxPC-3) as well as healthy pancreatic (HPNE) cells with various concentrations of exosomes. The effect of exosomal treatment on the epithelial-mesenchymal (E-M) transition of cancer cells was assessed by measuring the expression levels of E-cadherin and N-cadherin after 14 days of treatment. PTX was incorporated into the exosomes using ultrasonication, which involved five cycles of probe sonication at 50 W for 20 seconds, each followed by a 2-minute cooling interval to prevent thermal degradation and maintain vesicle integrity. In vitro cytotoxicity was evaluated in both 2D monolayer (PANC-1 and BxPC-3 cells) and 3D spheroid (PANC-1) models of pancreatic cancer.

Results: Exosomes were successfully isolated from the MSC conditioned media using the TFF method, resulting in a higher concentration compared to the traditional ultracentrifugation method. NTA results indicated that approximately 20,000 exosomes were isolated from each MSC. The mean hydrodynamic diameters of both the blank and PTX-loaded exosomes were 160 ± 16.4 nm and 154 ± 15.6 nm, respectively. Zeta potential measurements showed that the surface charges were -28.4 ± 2.2 mV for blank exosomes and -26.1 ± 1.99 mV for the loaded exosomes, indicating good colloidal stability. The presence of exosomal markers CD63 and CD81 confirmed that the isolated particles were indeed exosomes, while the expression of CD90 verified their origin from MSCs. Treatment of PANC-1 and BxPC-3 cancer cells with blank exosomes did not affect their viability during the study period. Additionally, the treatment did not alter the EMT of the PANC-1 and BxPC-3 cells. PTX was successfully incorporated into the exosomes, achieving a loading efficiency of 31.3 ± 1.93%. The IC50 values for PTX-loaded exosomes were 19.29 nM for PANC-1 cells and 7.53 nM for BxPC-3 cells, compared to 22.44 nM and 12.48 nM for free PTX, respectively. These results demonstrate that PTX delivered via MSC-derived exosomes exhibits enhanced antiproliferative effects, likely due to improved cellular uptake and drug bioavailability.

Conclusion: This study highlights the potential of exosomes derived from MSCs as an effective drug delivery system for PTX in pancreatic cancer. The exosomes were successfully isolated and loaded with paclitaxel, achieving a high loading efficiency. Furthermore, PTX-loaded exosomes showed enhanced antiproliferative effects compared to free PTX, with lower IC50 values observed in both the PANC-1 and BxPC-3 cell lines. Importantly, the MSC-derived exosomes did not alter the cadherin switching, which is a hallmark of EMT. This process is critical for cancer metastasis and tumor stemness. These findings highlight the promise of MSC-derived exosomes as a therapeutic platform for improving the targeting and efficacy of chemotherapeutic agents in pancreatic cancer treatment.

Acknowledgements: National Institute of General Medical Sciences (NIGMS) COBRE award 2P20 GM109024


Fig 1. Representative TEM images of (A) blank exosomes and (B) PTX-loaded exosomes; Size distribution of (C) blank exosomes and (D) PTX-loaded exosomes.


Fig 2. E-cadherin expression levels in (A) PANC-1 and (B) BxPC-3 cells; N-Cadherin expression levels in (C) PANC-1 and (D) BxPC-3 cells.


Fig 3. (A) Representative images of PANC-1 spheroids on Day 0 and Day 3. (B) Graphical representation of tumor volumes. Data represents mean ± SD (n=6).