Nanomedicine has witnessed remarkable progress, utilizing nano and microcarriers for treating diverse diseases. Enhancing the effectiveness of drugs through nanocarriers has been extensively researched in laboratories and clinical settings. Nanocarriers provide advantages such as protection of bioactive molecules, controlled release profiles, and targeted accumulation in specific tissues by leveraging enhanced permeability effects. However, achieving tissue-specific targeting often requires additional modifications to the carrier surface, increasing production costs and complexity. Moreover, the inherent tissue-specificity of carriers remains poorly understood and demands further exploration.

Many studies on nanocarriers rely on two-dimensional monolayer cell cultures, which do not faithfully replicate the complex three-dimensional networks present in the tumor microenvironment. Consequently, investigating carrier interactions within 2D cultures may yield unreliable results. To address this limitation, it is crucial to culture human cells in three dimensions, mimicking tissue-specific properties such as intercellular space, tissue stiffness, cell size, phagocytic function, receptor expression, and chemical gradients. Assessing the passage and uptake of nano and microcarriers within three-dimensional multicellular tumor spheroids can provide more accurate insights into the key parameters governing nanocarrier-cell interactions.

In this study, we explored the uptake and passage of polymeric carriers within cellular spheroids, with a focus on their behavior within complex three-dimensional cell systems. The carriers were constructed using a CaCO3 core and tannic acid/polyarginine-polyarginine/tannic acid. We investigated a wide range of carrier properties, including size (ranging from 300 to 1000 nm), charge (ranging from -19 to +24 mV), and stiffness (polymeric capsules and core-shell structures). To assess nanocarrier uptake and passage, we employed a combination of fluorescent microscopy and flow cytometry, enabling us to detect carriers in distinct zones of the cellular spheroids: the necrotic core, static zone, and proliferation zone, within 4T1 and L929 cell cultures. Through fluorescent microscopy, we determined carrier positions within normal and cancer spheroids, while flow cytometry facilitated the determination of their cellular and intercellular localization. Our innovative analytical method provided valuable insights into the essential aspects of carrier interactions within three-dimensional cellular systems. By utilizing three-dimensional cell cultures and employing comprehensive analytical techniques, our study significantly advances our understanding of polymeric carrier behavior within intricate cellular environments. The findings hold promising implications for the development of targeted drug delivery systems and pave the way for future advancements in the field of nanomedicine.