Together, this knowledge should facilitate the design of the next generation of CPPs. modulate the activity that these peptidic brokers take toward endosomal membranes and cytosolic egress. = 4, 5, 6, or 7 Arg residues) were synthesized with a universal N-terminal CK(TMR) scaffold. The charge density of the dfRn peptides remain relatively unchanged compared with dfTAT by truncating the length of the monomeric peptide (+3.7C4.4). To determine cytosolic penetration efficiency, the peptides were assayed at different concentrations [37]. The analogs with less guanidinium content (e.g., dfR4 and dfR5) yielded little to no cytosolic penetration activity at all of the tested concentrations tested. Yet, a drastic increase in cytosolic penetration activity (comparable to that of dfTAT) was observed for dfR6, dfR7, and dfR8. A threshold of 10 arginine residues is usually therefore required to achieve highly efficient cytosolic penetration. However, in constructs exceeding 12 total arginine residues (e.g., dfR7 and dfR8), increased cytotoxicity was observed. Taking into consideration both the cytosolic penetration and cytotoxicity data, 12 arginine residues (e.g., dfR6) represents a sweet spot for peptides to be highly efficient yet minimally cytotoxic. 4.2. Charge Density and Multimerization Play a Role in Successful Cytosolic Penetration While the dfRn peptides were generated to isolate the effect of guanidinium content, charge density and multimerization remained constant. A complementary study was conducted utilizing the nTAT series of peptides in which the number of TAT copies was modulated to evaluate the contribution of multimerization and charge density on cytosolic penetration efficiency [38]. The nTAT peptides (1TAT, 2TAT, and 3TAT) were generated by synthesizing a TMR-KGKGKG scaffold with one, two, or three copies of the TAT peptide conjugated to the -NH3+ of each lysine of the scaffold. Each peptide Efnb2 only differed in the number of TAT copies, which led to differences in charge and guanidinium density. When used to treat cells, 1TAT was incapable of entering cells. In contrast, 2TAT penetrated the cytosolic space of cells, albeit with only a modest efficiency. Moreover, 3TAT exhibited efficient cytosolic penetration after treating cells with as little as 1 M of peptide (Physique 3). Yet, while 1TAT and 2TAT were innocuous to cells, 3TAT was cytotoxic ( 10% cytotoxicity) when cells were treated with 5 M of peptide. When considering the cytosolic penetration and cytotoxicity data of the nTAT series in comparison with the dfRn series, there is a guanidinium threshold that is required to achieve efficient cytosolic penetration. As shown in Physique 2, this threshold enables the leaky fusion of BMP-containing lipid bilayers. Furthermore, these two datasets corroborate the idea that excessive arginine content leads to cytotoxicity. 4.3. Is There More to dfTAT Activity than Arginine Content? The results of the in vitro assays show that a threshold guanidinium content is required for membrane leakage. Notably, 2TAT is usually far less active than dfTAT or dfR6, even though these three peptides Glycyrrhizic acid meet the Arg threshold. The reason for this disparity in activity is not yet known. A difference between the peptides that could contribute to this disparity is the number of fluorophores that are incorporated into these constructs (one copy for 2TAT versus two copies for dfR6). In fact, a four to seven-fold decrease in cytosolic penetration and membrane lytic activity was observed for non-fluorescent variants of 2TAT and 3TAT. This suggests that the incorporation of fluorophore(s) enhances the cytosolic penetration activity of guanidinium-rich membrane lytic agents. Additionally, cell permeability is also affected by the how the guanidinium groups are displayed in a structure. For example, we have found that both R12 (a linear chain peptide of 12 arginine residues) and dfR6 are both cell-permeable. Yet, the linear representation (R12) leads to direct membrane translocation, whereas Glycyrrhizic acid the dimerized dfR6 undergoes endocytic-mediated cellular internalization (Figure 3). In particular, Glycyrrhizic acid we have observed that R12 translocates across the plasma membrane under conditions of membrane oxidation (e.g., when cells are grown at 20% oxygen, but not 2% oxygen, or when oxidants are present in growth media, but not when antioxidants are added) [36]. Therefore, linear peptides take a different route into.