Effects of particle uptake, encapsulation, and localization in cancer cells on intracellular applications

Highlights

• Endocytosis is affected by cell function, and likely malignancy.

• Particles endocytose into cells with reduced cell–cell interactions.

• Endocytosed particles may become unencapsulated by membranes with time.

• Endocytosis can safely be used to internalize particles for tracking.

Abstract

Endocytosis is a normal process in living cells, often used to internalize drug-containing particles and probes for intracellular mechanics. The cell type, and especially malignancy, may affect particle internalization and transport. Specifically, membrane-encapsulation following internalization can affect particle interaction with the cell interior. Hence, particle-tracking measurements that reveal intracellular mechanics and dynamics require determination of effects of encapsulation. Here, we compare closely related, breast-cancer cell lines with high- and low-metastatic potential (MP) and benign, control cells. We evaluate time-dependent particle internalization, localization with endocytotic-pathway organelles, and membrane encapsulation at 2, 6, 24, and 48 h after initial cell exposure to particles. High MP cells internalize particles more rapidly and in larger amounts than low MP and benign cells. Moreover, while only cells at the edge of two-dimensional colonies of benign cells internalized particles, all cancer cells uniformly internalize particles. Particles mostly colocalize with late endosomes (>80%), yet surprisingly, overall membrane encapsulation decreases with time, indicating release into the cytoplasm; encapsulation at 48 h is <35% in all three cell types. We discuss implications to drug delivery and show that encapsulation does not significantly affect intracellular particle-tracking experiments, showing the applicability of endocytosis.

Introduction

Increasing numbers of biomedical applications utilize nanoparticles internalized into living cells. Such particles have been used as vehicles for drug delivery [1], [2], [3], [4], [5], as sensors for imaging and diagnostics purposes [6], [7], and as probes to measure intracellular mechanics [8], [9], [10], [11]. In many of those assays, internalization of particles into cells is accomplished by the spontaneous mechanism of endocytosis. While endocytosis is a natural process in cells, there is typically no control over amounts of internalized particles or their localization within the cells. More precise control of particle amounts and localization can be obtained using other internalization methods, such as microinjection [12] and ballistic injection [13], [14]. Those internalization methods, however, also require specialized equipment and are invasive and typically damaging to the cells. Hence, endocytosis often remains a favored method, easily employed and minimally perturbing. Thus, to be able to rely on endocytosis for mechanical measurements, careful characterization is required to determine time-dependent amounts of internalized particles, their intracellular localization, and interactions with carrier organelles of the endocytotic pathway. That can affect mechanical measurements and optimal utilization of endocytosis for therapeutic purposes, and may reveal novel strategies for cancer therapy.

Fluid, molecules, and particles are naturally internalized when a sac, called an endosome, pinches off the cell membrane, engulfing and internalizing external objects [15]; that process may or may not be receptor mediated. From those early endosomes, cargo is typically transported into late endosomes that serve as a sorting station. Following sorting, cargo may be delivered into lysosomes for degradation or into the Golgi and endoplasmic reticulum (ER) for protein-relate processes. The cargo can also be released into the cell cytoplasm or discharged from the cell entirely (i.e. exocytosis); the encompassing vesicle is then recycled back into the plasma membrane. Those routes are normally used by cells for uptake of proteins and other macromolecules.

Synthetic particles have also been shown to undergo endocytosis, depending on their size, chemistry, and also cell type and activity. The effects of particle parameters such as size, shape, charge, and surface chemistry on endocytosis [16], [17], [18], [19], [20], [21], [22] and interaction with the cell interior [23] have been studied extensively. However, very few works have considered cell related parameters, such as cell type and cell cycle stage [24], [25]. Cell malignancy and metastatic potential (invasiveness) affect particle endocytosis [26], which has implications in drug delivery applications. Particle internalization has been shown to be slower in malignant breast cells than into their benign counterparts, yet more particles ultimately entered the malignant cells [27]. In addition, internalization of particles and their co-localization with lysosomes was faster in invasive, cancerous breast-cancer cells as compared to malignant (cancerous, yet non-invasive) breast tissue cells [28].

Here, we evaluate the time-dependent amounts of internalized 200-nm diameter particles and their membrane-encapsulation within endocytotic organelles, comparing benign, low metastatic potential (MP) and high MP epithelial breast cells. We note differences related to growth patterns and cell–cell interactions in two-dimensional (2D) culture. In addition, we determine the time-dependent endocytotic pathway of the particles by quantifying encapsulation in early and late endosomes, lysosomes, endoplasmic reticulum (ER), and the Golgi. Uptake and encapsulation dynamics were evaluated by determining colocalization of particles into each of the organelles at 2, 6, 24, and 48 h after exposure to particles. Our work shows uniform internalization of large numbers of particles into all the cancer cells, differing from the benign cells evaluated here. In addition, we show that particles gradually lose membrane encapsulation, and demonstrate that particle tracking experiments, to evaluate intracellular mechanics and dynamics, may be carried out after endocytosis.