(T0930-01-03) Intracellular Drug Delivery by Discrete Multiwalled Carbon Nanotubes for MDR Cancer Treatment

Purpose: Multi-drug resistance (MDR) is one of the toughest challenges in cancer treatment. A well-known cause of MDR involves the overexpression of efflux transporters, such as P-glycoprotein (P-gp) (1). Nanoparticles have shown promise in treating MDR in cancer treatment (2,3). Previously, we developed MMP-2 sensitive discrete multiwalled carbon nanotubes (dMWCNTs) that were efficient in targeting and killing tumor cells. In this study, we further extended the capability of dMWCNTs’ to deliver drugs intracellularly, target organelles, and overcome cancer MDR.

Methods: Doxorubicin hydrochloride (DOX), an anticancer drug as well as a P-gp substrate, was loaded onto dMWCNTs by overnight incubation at room temperature. Unloaded DOX was removed by dialysis (MWCO: 12 kDa) and the loading efficiency was calculated. To study the effect against MDR, the human uterine MDR cancer cell line, MES-SA/DX5 cells, was used as a model. DOX-loaded dMWCNTs were incubated with MES-SA/DX5 cells and the drug efflux was determined. Fluorescence microscopy and flow cytometry were used to visualize and quantify the intracellular DOX. To study the cell internalization mechanisms, the nanoparticles were incubated with the cells in different conditions (37 ℃ vs. 4 ℃) and in the presence of the endocytosis inhibitors chlorpromazine (for clathrin-mediated endocytosis), nystatin (for caveolin-mediated endocytosis), and dextran sulfate sodium (for micropinocytosis). The intracellular localization of the nanoparticles was analyzed by confocal microscopy. Organelle specific dyes were used to visualize the colocalization of dMWCNTs in mitochondria, endoplasmic reticulum (ER), golgi bodies, and cell membrane.

Results: Doxorubicin is loaded onto dMWCNTs through physical entrapment and π-π interactions, and the encapsulation efficiency was around 96%. The dMWCNTs were able to retain more than 60% of DOX in the cells for up to 30 hours after the removal of DOX treatment, while the retention for free DOX was only 35% under the same condition (Figure 1.A). Incubation of cells at 4 ℃ resulted in 97% reduction in uptake of dMWCNTs suggesting that the nanoparticles’ uptake is energy dependent. All the endocytosis inhibitors demonstrated dose-dependent reduction in cellular uptake suggesting that multiple pathways may be involved in nanoparticle uptake/endocytosis. At 2 hours upon internalization, the nanoparticles were mainly colocalized with ER (Figure 1.B). Some degree of colocalization was also evident in mitochondria.

Conclusion: In this study we were able to demonstrate dMWCNTs’ potential in the management of cancer’s MDR through their capability of intracellular drug delivery circumventing drug efflux. More excitingly, their significant localization in ER calls for sincere attention for further research in organelle-targeting. The data suggest that dMWCNTs may be an effective intracellular drug nanocarrier for MDR cancer treatment.

References: 1. Bukowski, K., Kciuk, M., and Kontek, R. Mechanisms of Multidrug Resistance in Cancer Chemotherapy. International journal of molecular sciences 2020 21(9), 3233.
2. Gavas, S., Quazi, S., and Karpiński, T.M. Nanoparticles for Cancer Therapy: Current Progress and Challenges. Nanoscale Res Lett 2021 16, 173
3. Wang, F., Wang, F., Dou, S., Xiong, M., Sun, T., and Wang, J., Doxorubicin-tethered responsive gold nanoparticles facilitate intracellular drug delivery for overcoming multidrug resistance in cancer cells. ACS Nano 2011 5 (5), 3679-3692


Figure 1. A) CNT/DOX is retained longer in MES-SA/DX5 cells compared to free DOX. X-axis refers to the time after treatment was removed, B) CNT/DOX shows strong colocalization with ER