Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Abstract

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather "difficult" or "hard to transfect," including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.

New generation of cell‐penetrating peptides: Functionality and potential clinical application

“…For example, noncovalent complexes formed from amphipathic CPPs and proteins could be easily transported into such difficult‐to‐transfect cells such as Kasumi‐1 and Leishmania cells 30–32 . However, we were unable to deliver plasmids into the same cells even when approaching the problem with a variety of different CPPs 33 . Because immune cells are also hardly to transfect cells it seems reasonable that other transport vectors are preferred for the development of vaccines against the novel coronavirus SARS‐CoV‐2.…”

Chiral Cyclobutane-Containing Cell-Penetrating Peptides as Selective Vectors for Anti-Leishmania Drug Delivery Systems

“…There are few examples of the use of CPP in the treatment of Leishmania [ 38 , 39 , 40 ]. Even so, the use of CPP as DDS on Leishmania parasites has been reported for the delivery of cargoes which are noncovalently complexed with CPP [ 41 , 42 , 43 ], or as covalent conjugates [ 44 , 45 , 46 , 47 , 48 ].…”

State of Art: Cell Penetration and Cell-Penetrating Peptides and Proteins

“…Electroporation is the method of choice for internalization of very large, conformational inflexible and charged molecules, DNA, pDNA and different kinds of RNA [102] . Thus, in our studies to transfect Leishmania tarentolae with recombinant DNA the electro-permeabilization remained the only reasonable tool [46]. Neither detergents and polymers nor magnetofection or CPPs are able to achieve transfection of these cells.…”

“…We detected a large difference between internalization of peptides and proteins conjugated to fluorescent markers or complexed with drugs on the one hand and linear and cyclic DNA on the other hand. In our hand's proteins, also very large proteins could be easily transported with CPPs, even into difficult to transfect cells [42,46]. Thus, non-covalent complexes formed from amphipathic CPPs and proteins could be easily transported into such difficult to transfect cells like Kasumi-cells and Leishmanias [11,42].…”

“…Thus, non-covalent complexes formed from amphipathic CPPs and proteins could be easily transported into such difficult to transfect cells like Kasumi-cells and Leishmanias [11,42]. On the other side we were unable to deliver plasmids into the same cells by approaching the use of a variety of different CPPs [46] Transduction and transfection of difficult-to-transfect cells seems to require rationally designed hybrid peptides, examples will be described in chapter. Composed peptides can promote the delivery of very large cargo proteins like CRISPR-Cas9 and -Cpf1 ribonucleoproteins into hardly to transfect human T-cell-derived Jurkat cells and primary NK cells [42].…”