Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

Abstract

Cell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This 'endosomal escape problem' has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

Keywords: TAT; cell-penetrating peptides; endocytosis; endosomal escape; protein transduction domains.

Figure 1

An overview of endocytosis. Endosome formation may be receptor (clathrin/caveolin) or non-receptor mediated (macropinocytosis). Following invagination (clatherin/cavaeolin) or extrvagination (macropinocytosis) of the plasma membrane, an early endosome is formed (1). As the endosome travels into the cell, it transitions to an early endosome and becomes increasingly acidified by pumping in cytosolic hydrogen ions into the vesicular lumen (2). As the early endosome matures, it begins to sort cargo by the formation of multiple smaller intracellular vesicles (ILVs) (3). Some of these vesicles may refuse with the endosomal membrane and deliver contents directly into the cytosol, called ‘backfusion’ while others will be destined for either the lysosome (4) or the trans-golgi network (TGN) (5). Cargo delivered to the lysosome will be degraded while cargo delivered to the TGN will be recycled back to the plasma membrane. Some receptors can bypass the TGN and be directly recycled back to the plasma membrane from the early endosome (6).

Figure 2

Proposed model for TAT:CAM-mediated intracellular delivery of CBS:CARGO. TAT fused to a calmodulin (TAT:CAM) will readily associate with cargo containing a calmodulin bind site (CBS:CARGO) in the extracellular environment owing to high levels of calcium (1). Following binding of TAT:CAM to a receptor, an endosome will form containing the TAT:CAM-CBS:CARGO as well as high levels of extracellular calcium. As the endosome matures, calcium will be pumped out of the intraluminal space while hydrogen ions are brought in (2). This shift in calcium concentrations within the endosome will cause the CBS:CARGO to disassociate from TAT:CAM, allowing for release of the cargo into the cytosol, presumably through IVLs, even if TAT:CAM remains tightly bound to its receptor (3). TAT:CAM itself may have multiple possible fates following formation of the late endosome. It may be sent to the lysosome for degradation if it retains its ability to bind its receptor in increasingly acidic conditions (4) or sent to the TGN (5) for recycling back to the plasma membrane (6).