In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.

The rapid emergence of multidrug-resistant pathogens has evolved into a global health problem as current treatment options are failing for infections caused by pan-resistant bacteria. Hence, novel antibiotics are in high demand, and for this reason antimicrobial peptides (AMPs) have attracted considerable interest, since they often show broad-spectrum activity, fast killing and high cell selectivity. However, the therapeutic potential of natural AMPs is limited by their short plasma half-life. Antimicrobial peptidomimetics mimic the structure and biological activity of AMPs, but display extended stability in the presence of biological matrices. In the present review, focus is on the developments reported in the last decade with respect to their design, synthesis, antimicrobial activity, cytotoxic side effects as well as their potential applications as anti-infective agents. Specifically, only peptidomimetics with a modular structure of residues connected via amide linkages will be discussed. These comprise the classes of α-peptoids (N-alkylated glycine oligomers), β-peptoids (N-alkylated β-alanine oligomers), β³-peptides, α/β³-peptides, α-peptide/β-peptoid hybrids, α/γ N-acylated N-aminoethylpeptides (AApeptides), and oligoacyllysines (OAKs). Such peptidomimetics are of particular interest due to their potent antimicrobial activity, versatile design, and convenient optimization via assembly by standard solid-phase procedures.

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 10⁵ cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide–cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered. Significance Bacterial drug resistance is a crucial threat to global health, and antimicrobials with novel mechanisms of action are urgently needed. Antimicrobial peptides are natural molecules that kill bacteria mostly by perturbing their membranes, and they constitute promising compounds for fighting resistant microbes. Their activity is normally tested under standardized conditions of bacterial density. However, the bacterial load in clinically relevant infections varies by many orders of magnitude. Here, we show that the minimum peptide concentration needed for bacterial growth inhibition can vary by more than 100-fold with an increase in the density of cells in the initial inoculum of the assay (a phenomenon termed the “inoculum effect”). These findings question the utility of the currently used activity screening assays.

The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.