Non-phagocytic eukaryotic cells can internalize particles <1 µm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50–1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50–500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diameter <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can determine the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 nm, and kinetic parameters may determine the almost exclusive internalization of such particles along this pathway rather than via caveolae.

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here we developed an analytical methodology, combining state-of-the-art molecular tools and correlative light and electron microscopy (CLEM), to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody-expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol.

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

Over the past decades, major efforts were undertaken to develop devices on a nanoscale level for the efficient and nontoxic delivery of molecules to tissues and cells, for the purpose of either diagnosis or treatment of disease. The application of such devices in drug delivery has proven to be beneficial for matters as diverse as drug solubility, drug targeting, controlled drug release, and transport of drugs across cellular barriers. Multiple nanotherapeutics have been approved for clinical treatment, and more products are being evaluated in preclinical and clinical trials. However, many biological barriers hinder the medical application of nanocarriers. There are two main classes of barriers that need to be overcome by drug nanocarriers: extracellular and intracellular barriers, both of which may capture and/or destroy therapeutics before they reach their target site. This Account discusses major biological barriers that are confronted by nanotherapeutics, following their systemic administration, focusing on cellular entry and endosomal escape of gene delivery vectors. The use of pH-responsive materials to overcome the endosomal barrier is addressed. Historically, cell biologists have studied the interaction between cells and pathogens in order to unveil the mechanisms of endocytosis and cell signaling. Meanwhile, it is becoming clear that cells may respond in similar ways to artificial drug delivery systems and, consequently, that knowledge on the cellular response against both pathogens and nanoparticulate systems will aid in the design of improved nanomedicine. A close collaboration between bioengineers and cell biologists will promote this development. At the same time, we have come to realize that tools that we use to study fundamental cellular processes, including metabolic inhibitors of endocytosis and overexpression/downregulation of proteins, may cause changes in cellular physiology. This calls for the implementation of refined methods to study nanocarrier–cell interactions, as is discussed in this Account. Finally, recent papers on the dynamics of cargo release from endosomes by means of live cell imaging have significantly advanced our understanding of the transfection process. They have initiated discussion (among others) on the limited number of endosomal escape events in transfection, and on the endosomal stage at which genetic cargo is most efficiently released. Advancements in imaging techniques, including super-resolution microscopy, in concert with techniques to label endogenous proteins and/or label proteins with synthetic fluorophores, will contribute to a more detailed understanding of nanocarrier-cell dynamics, which is imperative for the development of safe and efficient nanomedicine.

Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-beta-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas cell-association remained unaffected. These data strongly support the notion that complex internalization, rather than the direct translocation of plasmid across the plasma membrane, is a prerequisite for accomplishing effective lipoplex-mediated transfection. We demonstrate that internalized lipoplexes colocalize with transferrin in early endocytic compartments and that lipoplex internalization is inhibited in potassium-depleted cells and in cells overexpressing dominant negative Eps15 mutants. In conjunction with the notion that caveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.