(M0930-03-16) Paclitaxel Nanomicelles in 2D Monolayer and 3D Multicellular Spheroid Models of Triple-Negative Breast Cancer

Purpose: Conventional two-dimensional (2D) monolayer cell cultures fail to recapitulate the complex architecture and microenvironmental conditions characteristic of solid tumors. These cultures lack key features such as oxygen and nutrient diffusion gradients, extracellular matrix (ECM) interactions, and the multicellular organization necessary for physiologically relevant cell–cell communication. Consequently, their utility in accurately predicting therapeutic efficacy and tumor behavior is limited. In contrast, three-dimensional (3D) multicellular spheroids more closely emulate the structural and functional properties of in vivo tumor tissues, including the development of hypoxic cores, gradients in drug penetration, and increased resistance to apoptosis. Despite their limitations—particularly the absence of vasculature—3D spheroids offer a more representative in vitro model for studying tumor biology and drug response. The objective of this study was to evaluate and compare the effects of paclitaxel-loaded nanomicelles on cell viability and apoptosis in 2D monolayer versus 3D spheroid cultures of triple-negative breast cancer (TNBC) cell lines.

Methods: We used biodegradable amphiphilic PLGA-PEG block copolymers that self- assemble to design micelles and entrap the hydrophobic drug, paclitaxel, in its core. We characterized the nanomicles for its size, and drug loading. Effect of nanomicellar paclitaxel on cell death was studied in the triple negative breast cancer cell model, MDA-MB-231. The cell penetrating peptide iRGD was coadministered with the nanomicelles to study whether it enhanced the cellular uptake of the particles. iRGD works by binding to the overexpressed αvβ3 receptor on the cancer cell and vasculature surfaces, sensitizing the cell membrane to increase uptake of the nanomicelles. Therefore, it can potentially boost penetration of anticancer drugs deep into the tumor parenchyma and inhibit metastasis. In addition to standard cytotoxicity studies in 2D cell culture models, cytotoxicity and apoptosis were evaluated in 3D multicellular tumor spheroids.

Results: Paclitaxel was successfully encapsulated into nanomicelles, achieving a loading efficiency of 55%, as determined by high-performance liquid chromatography (HPLC). There was no statistically significant difference in particle size between paclitaxel-loaded and blank micelles; both exhibited an average diameter of approximately 75 nm. Cytotoxicity studies using the Glo assay demonstrated that 3D tumor spheroid models exhibited significantly greater resistance to paclitaxel compared to 2D monolayer cultures, with a ~700-fold increase in IC₅₀ values observed in the 3D system. In 2D cultures, micellar paclitaxel induced significantly higher cell death compared to free paclitaxel (Figure 1); however, this enhanced cytotoxicity was not observed in the 3D spheroid model. Co-administration of iRGD resulted in synergistic growth inhibition in both 2D and 3D cultures (Figure 1). Apoptosis was quantified using flow cytometry with the Guava Nexin assay, which utilizes Annexin V-PE and 7-aminoactinomycin D (7-AAD) staining. Flow cytometric analysis indicated a higher percentage of apoptotic cells in 2D cultures at lower drug concentrations, with or without micellar formulation and/or iRGD co-treatment, compared to the 3D spheroid model (Figure 2).
All data were analyzed using statistical methods to determine significance.

Conclusion: The therapeutic efficacy of paclitaxel-loaded micelles was demonstrated in 2D monolayer cultures, where they induced significantly greater cell death compared to equivalent concentrations of free paclitaxel. Co-administration with the tumor-penetrating peptide iRGD further enhanced cytotoxicity in the 2D model. While paclitaxel micelles alone did not elicit comparable levels of cytotoxicity in 3D spheroid cultures, their combination with iRGD resulted in additive effects, suggesting improved penetration and distribution of the micellar formulation within the tumor-like architecture of the spheroids.
These findings support two key conclusions: (1) combination therapy using paclitaxel-loaded micelles and iRGD holds potential as a clinically relevant strategy for enhanced targeting and treatment of solid tumors in vivo, and (2) the 3D spheroid model of triple-negative breast cancer (TNBC) exhibits substantially higher drug resistance relative to 2D monolayers, which may partially explain the limited translational success of therapies optimized solely in 2D systems.

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Fig 1 – Comparative studies of IC50 in 2D (yellow) and 3D (terracotta), X-axis normalized TNBC cell viability, Y-axis treatments with concentrations


Fig 2: Comparative apoptosis study using flow-cytometry