Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins.

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the "histidine switch" principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes-including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase-into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Extracellular Metabolites of Heterotrophic Auxenochlorella protothecoides: A New Source of Bio-Stimulants for Higher Plants.

The biodiversity of microalgal species is enormous, and their versatile metabolism produces a wide diversity of compounds that can be used in food, healthcare, and other applications. Microalgae are also a potential source of bio-stimulants that enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits. In this study, the extracellular metabolites of Auxenochlorella protothecoides (EAp) were prepared using three different culture strategies, and their effects on plant growth were examined. Furthermore, the composition of EAp was analyzed by GC-MS. The elongation of lateral roots and the cold-tolerance of Arabidopsis thaliana and Nicotiana benthamiana were promoted by EAp. Moreover, EAp from high-cell-density fermentation stimulated the growth of the leafy vegetables Brassica rapa and Lactuca sativa at dilutions as high as 500- and 1000-fold. Three major groups of compounds were identified by GC-MS, including organic acids or organic acid esters, phenols, and saccharides. Some of these compounds have known plant-stimulating effects, while the rest requires further investigation in the future. Our study demonstrates that EAp is a potential bio-stimulant, while also providing an environmentally friendly and economical microalgae fermentation process.