The Clostridium botulinum C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells.

The binary Clostridium (C.) botulinum C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA63. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins' catalytic A subunits. C2IIa as well as PA63 deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA63, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA63. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency.

Analysis of Triterpenoid Saponins Reveals Insights into Structural Features Associated with Potent Protein Drug Enhancement Effects.

Plant-based saponins are amphipathic glycosides composed of a hydrophobic aglycone backbone covalently bound to one or more hydrophilic sugar moieties. Recently, the endosomal escape activity of triterpenoid saponins has been investigated as a potentially powerful tool for improved cytosolic penetration of protein drugs internalized by endocytic uptake, thereby greatly enhancing their pharmacological effects. However, only a few saponins have been studied, and the paucity in understanding the structure-activity relationship of saponins imposes significant limitations on their applications. To address this knowledge gap, 12 triterpenoid saponins with diverse structural side chains were screened for their utility as endosomolytic agents. These compounds were used in combination with a toxin (MAP30-HBP) comprising a type I ribosome-inactivating protein fused to a cell-penetrating peptide. Suitability of saponins as endosomolytic agents was assessed on the basis of cytotoxicity, endosomal escape promotion, and synergistic effects on toxins. Five saponins showed strong endosomal escape activity, enhancing MAP30-HBP cytotoxicity by more than 106 to 109 folds. These saponins also enhanced the apoptotic effect of MAP30-HBP in a pH-dependent manner. Additionally, growth inhibition of MAP30-HBP-treated SMMC-7721 cells was greater than that of similarly treated HeLa cells, suggesting that saponin-mediated endosomolytic effect is likely to be cell-specific. Furthermore, the structural features and hydrophobicity of the sugar side chains were analyzed to draw correlations with endosomal escape activity and derive predictive rules, thus providing new insights into structure-activity relationships of saponins. This study revealed new saponins that can potentially be exploited as efficient cytosolic delivery reagents for improved therapeutic drug effects.

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies.

Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed.

Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers.

The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

Endolysosomal environment-responsive photodynamic nanocarrier to enhance cytosolic drug delivery via photosensitizer-mediated membrane disruption.

The endolysosome is a major barrier for the effective intracellular delivery by conventional nanocarriers. Herein, we demonstrate that endolysosome environment-responsive photodynamic nanocarriers (EPNs) are capable of encapsulation of the hydrophobic drug paclitaxel (PTX) and photosensitizer (PS)-mediated ELB disruption for effective cancer therapy. EPNs were self-assembled from PS (chlorin e6, Ce6) or Black Hole Quencher-3 (BHQ3) conjugated covalently to polypeptide-based amphiphilic copolymers [monomethoxy polyethylene glycol-block-poly(ß-benzyl-L-aspartic acid), mPEG-pBLA]. EPNs have a spherical shape and a unimodal size distribution below 100 nm. Photoquenching of the EPNs was dependent on the molar ratio of mPEG-pBLA-BHQ3/mPEG-pBLA-Ce6. However, in the presence of the endolysosomal enzyme (e.g., esterase), the benzyl ester bond is cleaved which leads to the structural collapse of EPNs, thus triggering drug release and restoring photoactivity. Live cell imaging studies demonstrated that PS-mediated lipid peroxidation significantly increased the ability of model drug (i.e., Nile red) to overcome the ELB. In comparison with PTX treatment alone, the combined treatment of PTX encapsulated EPNs with laser irradiation synergistically induced the death of HeLa and drug-resistant HCT-8 cells in vitro, and suppressed CT-26 tumor growth in vivo. These results suggest that this approach is a promising platform for cancer treatment. Furthermore, this EPN system offers significant potential for effective cytosolic delivery of chemical and biological therapeutics.