Alterations in Cellular Processes Involving Vesicular Trafficking and Implications in Drug Delivery

Abstract

Endocytosis and vesicular trafficking are cellular processes that regulate numerous functions required to sustain life. From a translational perspective, they offer avenues to improve the access of therapeutic drugs across cellular barriers that separate body compartments and into diseased cells. However, the fact that many factors have the potential to alter these routes, impacting our ability to effectively exploit them, is often overlooked. Altered vesicular transport may arise from the molecular defects underlying the pathological syndrome which we aim to treat, the activity of the drugs being used, or side effects derived from the drug carriers employed. In addition, most cellular models currently available do not properly reflect key physiological parameters of the biological environment in the body, hindering translational progress. This article offers a critical overview of these topics, discussing current achievements, limitations and future perspectives on the use of vesicular transport for drug delivery applications.

Keywords: cellular vesicles; disease effects on vesicular trafficking; drug delivery systems and nanomedicines; drug effects on vesicular trafficking; fission and intracellular trafficking; role of the biological environment; transcytosis and endocytosis of drugs carriers; vesicle fusion.

Figure 1

Endocytic pathways. Endocytosis via membranous vesicles encompasses several mechanisms where the plasmalemma internalizes objects (phagocytosis) and extracellular fluid (pinocytosis) in intracellular vesicles. Some vesicles concentrate receptors that mediate uptake of specific ligands via receptor-mediated endocytosis (marked by *), while others (e.g., macropinocytosis) largely internalize fluid and solutes by non-specific means. Pinocytosis is subdivided in macro- and micropinocytosis depending on the size of the vesicles that form and the latter can be mediated by classical (clathrin- and caveolae-mediated pathways) or non-classical (clathrin and caveolae independent) routes, all of which can be classified as per their dynamin dependence (marked by ^#). Apart from the trafficking destinations shown, some markers (e.g., platelet–endothelial cell adhesion molecule 1 (PECAM-1) and intercellular adhesion molecule 1 (ICAM-1) associated with the cell adhesion molecule (CAM)-mediated pathway) may shuttle back and forth between the cell surface and a subplasmalemma vesicular compartment whose membrane is continuous with the plasmalemma. Adapted and reproduced with permission from Figure 12.2 in [26]. Copyright 2016 Pan Stanford.

Figure 2

Vesicular transport. Intracellular vesicles transport cargo between the plasmalemma and organelles or from an organelle to another, which encompasses the processes of: (1) budding of a nascent vesicle from the donor membrane and pinching off into the cytosol; (2) trafficking aided by cytoskeletal elements; (3) tethering of the vesicle to the acceptor membrane; and (4) fusion to deliver cargo to the acceptor compartment. Adapted and reproduced with permission from Figure 3.2 in [2]. Copyright 2016 Pan Stanford.

Figure 3

Endocytic alterations caused by disease. (A) Micrographs (top panels) and image quantification (bottom graphs) of the uptake of transferrin (Tf; left side) via clathrin-mediated endocytosis (CME) or cholera toxin B (CTB; right side) via caveolae-mediated endocytosis (cavME) in fibroblasts from wild-type (Wt) individuals or patients of Niemann–Pick type A (NPD), Niemann–Pick type C (NPC), Gaucher and Fabry diseases. Green: internalized ligand; yellow-red:cell surface-bound ligand; dashed lines: cell borders; scale bar: 10 μm. * p < 0.05, Student’s t test. (B) Endocytosis time constants after short (50 action potentials) or long (300 action potentials) stimulation of cortical neurons from Wt mice vs. mice expressing Parkinson-like R258Q mutation in synaptojanin 1 (SJ1), a molecule involved in synapse endocytic signaling. **** p < 0.00001, Mann–Whitney U test. (C) Uptake of endocytic markers in HBEC30KT normal cells vs. Hcc4017 cancer cells from the same patient. C-Cav-IE: Clathrin- and caveolae-independent endocytosis. *** p < 0.0005, Student’s t test. Data are mean ± standard error of the mean (SEM) for (A) and (B), and standard deviation (SD) for (C). Adapted and reproduced with permission from: (A) Figures 4 and 5 in [94]; (B) Figure 5B in [95]. Copyright 2017 Elsevier Inc.; (C) Figure 2A in [96]. Copyright 2015 American Association for Cancer Research.

Figure 4

Endocytic alterations caused by therapeutic drugs. (A) Western blot protein bands (upper panels) and densitometry (bottom panels) showing the effect of the antimalarial and cancer treatment drug, chloroquine, on the level of vesicular transport elements in PC12 cells activated for endocytosis with cholera toxin B. (B) The upper graph shows lysosomal exocytosis, measured as extracellular release of lysosomal enzyme HEXB, in fibroblasts from normal individuals treated with by δ- or α-tocopherol. The bottom panel shows Western blot analysis of flotillin-2 in exosomal fraction of cell treated with hydroxypropyl-β-cyclodextrin (a positive control), δ-tocopherol, or ionomycin, normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. Data are mean ± SEM, ** p < 0.05, Student’s t test. Adapted and reproduced with permission from: (A) Figure 2 in [139]. Copyright 2006 Federation of European Biochemical Societies; (B) Figure4 in [140]. Copyright 2012 The American Society for Biochemistry and Molecular Biology, Inc.

Figure 5

Role of carrier size and shape in the cellular uptake and trafficking via intercellular adhesion molecule 1 (ICAM-1). (A) Kinetics of endocytosis of spherical, ICAM-1-targeted polymer particles of various sizes by endothelial cells in culture. (B) Kinetics of uptake of micrometer-range size, ICAM-1-targeted polymer particles of spherical vs. elongated-disc shape. (C) Kinetics of lysosomal trafficking of said spherical vs. elongated polymer particles, also comparing nano- vs. micrometer-size range. Data are mean ± SEM. Adapted and reproduced with permission from Figures 4B and 6B in [184]. Copyright 2008 The American Society of Gene Therapy.

Figure 6

Endocytic alterations caused by drug carriers. (A) Kinetics of endocytosis of via intercellular adhesion molecule 1 (ICAM-1)-targeted polymer nanocarriers (anti-ICAM/NCs) by HUVEC cells (traced by fluorescence microscopy) and that of cell surface levels of ICAM-1 receptor during nanocarrier uptake (traced by radioactive labeling). (B) Relative level of binding and endocytosis of anti-ICAM/NCs applied as a second dose to HUVEC cells (and traced by fluorescence microscopy), either 30 min or 3 h after a first dose of NCs. * p < 0.05, Student’s t test. (C) Relative level of binding and endocytosis of anti-ICAM/NCs applied as a second dose to HUVEC cells (and traced by fluorescence microscopy), either 30 min or 3 h after a first dose of NCs. (C) Lung targeting, expressed as % injected dose per gram of tissue (%ID/g; radioactive tracing), of a first dose of anti-ICAM/NCs injected i.v. in mice vs. that of NCs applied as a second dose 15, 30, or 150 min after the first dose. Control non-specific IgG/NCs are also shown. Data are mean ± SEM, compared by Student’s t test. Adapted and reproduced with permission from Figures 1 and 3 in [64]. Copyright 2005 The American Sociaty of Hematology.