Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Insight into the Mechanism of Internalization of the Cell-Penetrating Carrier Peptide Pep-1 by Conformational Analysis

Different secondary structures of the pep-1 protein were blamed for transmembrane internalization process of drugs and drug deliveries. But which structure will be important for transmembrane delivery was still not clear. In this study, interactions between pep-1 and cell membranes were studied. Pep-1 in the buffer (Pep-1) and pep-1 on graphene (PDS/G) or they on graphene oxide (PDS/GO) were composed as the transmembrane delivery system to study the different secondary structure of pep-1 that influence for their transmembrane delivery. The curves of chirascan circular dichroism (CD) and all-atom discontinuous molecular dynamics (DMD) simulations illuminate that, in a buffer environment, most pep-1 formed 3–10 helix structures. Meanwhile, when Pep-1 composed graphene slice and formed PDS/G, 3–10 helix and alpha-helix structures can be found in small quantities. When they on graphene oxide and formed PDS/GO, coil or type II beta-turn structure can be found from most of the pep-1 and 3–10 helix structure disappeared. By using sum-frequency generation (SFG) vibrational spectroscopy, we found that pep-1 with 3–10 helix structures in buffer solutions damaged the lipid bilayer violently. PDS/G with less 3–10 helix structures will change the orientation of lipid bilayer effectively but slightly. Pep-1 with coil or type II Beta-turn in PDS/GO cannot influence the structure of lipid bilayers. Hemolysis experiments also proved that when pep-1 composed as PDS/G, they will change the orientation of the plasma membrane of red blood cells effectively but slightly. When they attach on the GO and formed PDS/GO, the plasma membrane of red blood cells cannot be influenced. In conclusion, 3–10 helix structures will be positively correlated with disturbance of membranes. These results will be effectively guided the clinic application of pep-1 as a transporter of the drug delivery system.

Formulation and Characterization of Polyurethane Microstructures with Propolis Extract

Propolis is a natural apicultural product derived from plant resins with impressive health benefits. Its major biologically active substances are barely soluble in water, but this could be increased by proper formulations.The aim of the present study was to obtain a transmembrane delivery system based on polyurethane microstructures for eight propolis samples collected from different regions of Western Romania and to characterize them by differential scanning calorimetry (DSC); the assessed parameters were the particle size, the polydispersity index and their clustering tendency. Polyurethane polymers based on isophorone-diisocyanate and polyethylene glycol 200/ethylene glycol were chosen as carriers for the propolis extract. A perfect inclusion of propolis inside the structures was observed for half of the samples. Particle size ranged between 504 and 621 nm and was confirmed by the low stability against aggregation (Zeta potential: 16.3-19.8 mV). The polydispersity index was between 0.4 and 0.7.

Synthesis and Physico-Chemical Evaluation of Polyurethane Microstructures for Transmembrane Delivery of Reynoutria japonica Extract

Polymer microstructures containing plant extracts are a novel technique used to increase the bioavailability of active agents with low aqueous solubility. Japanese knotweed (Reynoutria japonica) is an invasive plant with multiple biomedical properties. The aim of the present study was to obtain and characterize polyurethane microstructures used as a drug delivery system of a Japanese knotweed extract. The results indicate the obtaining of microstructures with an almost neutral pH, with sizes between 540 and 1134 nm, with a medium stability against the tendency to form clusters and a very good thermal stability. These first results present the potential of the obtained polyurethane microstructures as candidates for the transmembrane drug delivery of Japanese knotweed extracts, as a starting point for further bioactivity investigations.