(T1430-06-34) Improving Doxorubicin’s Tumor Penetration and Retention by Discrete Multiwalled Carbon Nanotube-Based Drug Delivery

Purpose: Cancer treatment remains an elusive target given the complexity of the disease and shortcomings of current medications. Some of the major challenges such as poor solubility, off-target toxicity, poor tumor penetration, and multi-drug resistance (MDR) can be overcome through nanoparticle-based drug delivery systems¹. Doxorubicin (DOX) is a widely used anticancer drug restricted by its non-specific toxicity, susceptibility to MDR, and poor tumor penetration and retention². We have been successful in loading DOX into our discrete multiwalled carbon nanotubes (dMWCNTs). The dMWCNTs/DOX were found to be significantly less susceptible to efflux than DOX in MDR cancer cell lines (MES/SA-DX5 and NCI/ADR-RES). In this study, we have explored the behavior of the dMWCNTs in tumor tissues using a 3D cell spheroid model as well as an orthotropic breast tumor (4T1) mouse model.

Methods: To load DOX, the dMWCNTs were incubated with DOX solution overnight at room temperature. Unloaded DOX was separated by Nanosep centrifugal filter (MWCO=300kD) by centrifuging at 14000 rpm for 20 minutes. The filtrate was analyzed by LC-MS to determine the amount of unloaded DOX, and the encapsulation efficiency was calculated. The 4T1 cell spheroids were used as a model to mimic the complexity of tumor in vitro. To study the tumor penetration, dMWCNTs/DOX and DOX were incubated with the spheroids for 2 hours. A confocal microscope was used to visualize the DOX fluorescence in the spheroid at a step size of 20 µm along the Z-axis. For tumor retention study, an orthotropic breast tumor (4T1) model was established in the BALB/c mouse. After 3 weeks of tumor implantation, dMWCNTs/DOX and DOX were injected intratumorally. Tumors were collected 16 hours later, homogeneized, and fluorescence of the homogenate was measured using a plate reader. The role of endo/lysosomes in the uptake of dMWCNTs in NCI/ADR-RES cells was assessed by using rhodamine-PE (Rh-PE) as a fluorescent probe. The co-localization of the dMWCNTs/Rh-PE with endo/lysosomes stained by LysoTracker Green was observed using confocal microscope. Finally, to determine if the release of DOX from the endocytosed dMWCNTs/DOX is facilitated by the acidic pH of endo/lysosomes, a drug release study was carried out at different pHs: 5.5 and 7.4 for 24 hours.

Results: About 97% of DOX was loaded onto the dMWCNTs. The tight spheroids could be formed in one week and had a diameter of 644 ± 62 µm. Compared to DOX, dMWCNTs/DOX penetrated the spheroid better, as evidenced by their stronger fluorescence in deeper regions (Figure 1). Following the intratumor injection, it was found that the retention of dMWCNTs/DOX was almost 100% higher than that of DOX (Figure 2). The co-localization study demonstrated that free Rh-PE was not endocytosed but dMWCNTs/Rh-PE had a significant association with endo/lysosomes (Figure 3). The release of DOX from dMWCNTs/DOX was found to be pH-dependent. At 24 hours, the cumulative DOX release at pH 5.5 was > 50% higher than that at pH 7.4.

Conclusion: Our dMWCNTs proved to be an excellent carrier for DOX. The pi-pi stacking between DOX and dMWCNTs results in strong adsorption of DOX ³. Compared to the 2D cell monolayers, 3D cell spheroids offer more resistance to drug molecules ⁴ which was evident with DOX. On the other hand, dMWCNTs were able to penetrate deeper into the spheroids, making them an attractive delivery platform to overcome DOX resistance. Once inside a tumor, dMWCNTs stay there longer as opposed to free DOX which is cleared readily. The uptake of dMWCNTs via endocytosis and the elevated DOX release at acidic pH suggest that dMWCNTs can hold DOX during in vivo delivery at physiologic pH but tend to release the loaded drug intracellularly at tumor site. These impressive performances of dMWCNTs at tissue level corroborate our previous findings in cell-based assays. In future, the in vivo anticancer study would further consolidate dMWCNTs’ prospect as an effective nanocarrier for cancer drug delivery. Surface engineering with targeting ligands or stimuli-sensitive moieties ⁵ would further improve dMWCNTs’ tumor specificity for drug delivery.

References: (1) Zhu, L.; Wang, T.; Perche, F.; Taigind, A.; Torchilin, V. P. Enhanced Anticancer Activity of Nanopreparation Containing an Mmp2-Sensitive Peg-Drug Conjugate and Cell-Penetrating Moiety. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 17047.
(2) Lim, J. X.; Yong, Y. K.; Dewi, F. R. P.; Chan, S. Y.; Lim, V. Nanoscale strategies: doxorubicin resistance challenges and enhancing cancer therapy with advanced nanotechnological approaches. Drug Delivery and Translational Research 2025. DOI: 10.1007/s13346-025-01790-3.
(3) Yaghoubi, A.; Ramazani, A. Anticancer DOX delivery system based on CNTs: Functionalization, targeting and novel technologies. J Control Release 2020, 327, 198-224. DOI: 10.1016/j.jconrel.2020.08.001 From NLM.
(4) Świerczewska, M.; Sterzyńska, K.; Ruciński, M.; Andrzejewska, M.; Nowicki, M.; Januchowski, R. The response and resistance to drugs in ovarian cancer cell lines in 2D monolayers and 3D spheroids. Biomedicine & Pharmacotherapy 2023, 165, 115152. DOI: https://doi.org/10.1016/j.biopha.2023.115152.
(5) Yao, Q.; Dai, Z.; Hoon Choi, J.; Kim, D.; Zhu, L. Building Stable MMP2-Responsive Multifunctional Polymeric Micelles by an All-in-One Polymer–Lipid Conjugate for Tumor-Targeted Intracellular Drug Delivery. ACS Applied Materials & Interfaces 2017, 9 (38), 32520-32533. DOI: 10.1021/acsami.7b09511.


Figure 1. Tissue penetration study: a) Z-stacks of dMWCNTs/DOX and DOX treated 4T1 spheroids, and b) Intensity profile of spheroid at the stack 40µm from the surface shows that dMWCNTs/DOX have higher penetration than DOX.


Figure 2. Co-localization assay: dMWCNTs/Rh-PE (red) co-localization with endo/lysosomes (green) confirmed by the formation of yellow dots.


Figure 3. Tumor retention study: DOX fluorescence in the tumor homogenate measured by fluorometer shows that the DOX retention was almost double in the tumor treated with dMWCNTs/DOX than the tumor treated with the DOX solution.