Studies of cell-penetrating peptides by biophysical methods

Abstract

Biophysical studies have a very high impact on the understanding of internalization, molecular mechanisms, interactions, and localization of CPPs and CPP/cargo conjugates in live cells or in vivo. Biophysical studies are often first carried out in test-tube set-ups or in vitro, leading to the complicated in vivo systems. This review describes recent studies of CPP internalization, mechanisms, and localization. The multiple methods in these studies reveal different novel and important aspects and define the rules for CPP mechanisms, hopefully leading to their improved applicability to novel and safe therapies.

Confocal Laser Scanning Microscopy and Model Membranes to Study Translocation Mechanisms of Membrane Active Peptides

“…In this case, a very strong accumulation of peptides on membranes was observed. Additional to the values of intensity measured inside GUVs, the internalization of peptides can be demonstrated by their accumulation in internal membranes of multivesicular GUVs, as can be observed for NKCS- [15][16][17][18][19][20][21][22][23][24][25][26][27] in Figure 4B. The same multivesicular GUVs indicated no lytic effect induced by the peptide, as well.…”

“…The lytic effect was observed by the efflux of AF 488 for TAT and NKCS, indicating the pore mechanism of internalization for these peptides (see Figure 3). The NKCS- [15][16][17][18][19][20][21][22][23][24][25][26][27] and tLyP-1 peptides internalized without the release of AF 488, suggesting a direct translocation mechanism. In neutral membranes comprised of POPC and cholesterol, peptides neither internalize nor induce the release of the internal fluorescent tracer Alexa Fluor 488, regardless of the structure, even after 30 min of incubation, as it can be observed in Figure 2.…”

“…NKCS, NKCS- [15][16][17][18][19][20][21][22][23][24][25][26][27] and tLyP-1 peptides labeled with the fluorescent molecule Dy647P1 (Dynomics) were synthesized by Biosyntan (Berlin, Germany) in purity >95%. The fluorescent dye was coupled to the C-terminus through an additional cysteine as maleimide.…”

“…In anionic membranes, with 30 mol% POPS, a similar effect to POPE was observed (Figure 4). TAT, NKCS, NKCS- [15][16][17][18][19][20][21][22][23][24][25][26][27] and tLyP-1 peptides were internalized in these GUVs, but only the first two were able to form pores. In this case, a very strong accumulation of peptides on membranes was observed.…”

“…This peptide enters the GUVs mimicking the cytoplasmic membrane of bacteria by a pore mechanism as well [10]. NKCS- [15][16][17][18][19][20][21][22][23][24][25][26][27] is a derivative of NKCS, corresponding to the region of C-terminal fragment from residue 15 to 27. tLyP-1 is a tumor homing peptide, Pharmaceutics 2022, 14, 1699 3 of 10 the truncated linear form of cyclic peptide LyP-1. tLyP binds to the neuropilin (NRP1 and/or NRP2) receptor overexpressed in glioma, breast cancer, prostate cancer and tumor vasculature [10,11].…”