Cell-Penetrating Peptides, Electroporation, and Drug Delivery

Cell-Penetrating Peptides, Electroporation, and Drug Delivery Kevin Cahill May 30, 2022 cahill@unm.edu Biophysics Group, Department of Physics & Astronomy, University of New Mexico, Albuquerque, NM 87131 ABSTRACT Certain short polycations, such as TAT and oligoarginine, rapidly pass through the plasma membranes of mammalian cells by a mechanism called transduction, as well as by endocytosis and macropinocytosis. These cell-penetrating peptides (CPPs) can carry with them cargos of 30 amino acids, more than the nominal limit of 500 Da and enough to be therapeutic. An analysis of the electrostatics of a charge outside the cell membrane and some recent experiments suggest that transduction may proceed by molecular electroporation. Ways to target diseased cells, rather than all cells, are discussed. I. THE PROBLEM OF DRUG DELIVERY We could cure cancer if we knew how to deliver a drug intact to the cytosol of every cancer cell, sparing healthy cells. The circulatory system can deliver a drug to every cell in the body, and certain chemical tricks protect drugs from peptidases [1] and nucleases [2]. But it’s harder to cope with antibodies, spare healthy tissues, and get drugs past the plasma membrane, which blocks or endocytoses molecules in excess of 500 Da [3]. This paper is about cell-penetrating peptides and other cations that can overcome the 500-Da restriction barrier and about tricks that may spare healthy cells. Section II describes several cell-penetrating peptides (CPPs), and section III sketches a variety of therapeutic applications of CPPs. Section IV reviews basic facts about the lipid bilayer of the eukaryotic cell. New work on the electro- statics of the bilayer is presented in section V. Section VI sketches a model [4, 5] of the transduction of polyargi- nine and mentions some experimental support [6] which the model has recently received. Section VII discusses a broader class of cell-penetrating molecules and suggests ways to target cancer cells. II. CELL-PENETRATING PEPTIDES In 1988, two groups [7, 8] working on HIV reported that the trans-activating transcriptional activator (TAT) of HIV-1 can cross cell membranes. The engine driving this 86-aa cell-penetrating peptide (CPP) is residues 48– 57 grkkrrqrrr which carry a charge of +8e. Other CPPs were soon found. Antp (aka Penetratin, PEN) is residues 43–58 rqikiwfqnrrmkwkk of Antennape- dia, a homeodomain of the fly; it carries a charge of +7e. The polyarginine Rn carries charge +ne, where often n = 7, 8, or 9. Other CPPs have been discov- ered (VP22) or synthesized (transportan). The struc- tural protein VP22 of the tegument of herpes simplex virus type 1 (HSV-1) has charge +15e. Transportan gwtlnsagyllg-k-inlkalaalakkil-amide is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan and a connecting lysine [9]. With its ter- minal amide group, its charge is +5e. These and other short, positively charged peptides can penetrate the plasma membranes of live cells and can tow along with them cargoes that greatly exceed the 500 Da restriction barrier. They are promising therapeutic tools when towing cleverly chosen peptide cargoes of from 8 to 33 amino acids [10–22]. Many early experiments on CPPs were wrong be- cause the cells were fixed or insufficiently washed. Even careful experiments sometimes have yielded inconsistent results—in part because fluorescence varies with the (sub)cellular conditions and the fluorophores [23]. Yet some clarity is emerging: TAT carries cargoes across cell membranes with high efficiency by at least two functionally distinct mechanisms according to whether the cargo is big or small [24]. Big cargoes, such as pro- teins or quantum dots, enter via caveolae endocytosis and macropinocytosis [25, 26], and relatively few escape the cytoplasmic vesicles in which they then are trapped [24]. Small cargoes, such as peptides of fewer than 30– 40 amino acids, enter both slowly by endocytosis and rapidly by transduction with direct access to the cytosol, an unknown mechanism that uses the membrane poten- tial [24, 27–30]. Peptides fused to TAT enter cells within seconds [31]. It remains unclear how big cargoes aided by several CPPs enter cells [32]. For instance, superparamagnetic nanoparticles encased in aminated dextran and attached to 45 tat peptides are thought to enter cells by adsorptive endocytosis[33–35] but they do enter slowly at 4◦C [36]. III. THERAPEUTIC APPLICATIONS The use of a cell-penetrating peptide (CPP) to carry into cells a biologically active peptide of up to some 35 amino acids (aa) allows for 2035 = 3 × 1045 different peptides and may lead to strikingly smart drugs, some of which may selectively target tumor cells [10]. I will now briefly describe 11 promising advances toward this goal of cell-penetrating-peptide drugs (CPPDs). The CXC chemokine receptor 4 (CXCR4) is over- expressed in > 20 types of cancer, including prostate, breast, colon,

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