Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78.

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa). The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition. We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.

A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells.

Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78.

Killing of Mycobacterium avium by lactoferricin peptides: improved activity of arginine- and D-amino-acid-containing molecules.

Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections.

Comparison of the efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery.

The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

Hydration water and peptide dynamics--two sides of a coin. A neutron scattering and adiabatic calorimetry study at low hydration and cryogenic temperatures.

In the present work we bridge neutron scattering and calorimetry in the study of a low-hydration sample of a 15-residue hybrid peptide from cecropin and mellitin CA(1-7)M(2-9) of proven antimicrobial activity. Quasielastic and low-frequency inelastic neutron spectra were measured at defined hydration levels - a nominally 'dry' sample (specific residual hydration h = 0.060 g/g), a H2O-hydrated (h = 0.49) and a D2O-hydrated one (h = 0.51). Averaged mean square proton mobilities were derived over a large temperature range (50-300 K) and the vibrational density of states (VDOS) were evaluated for the hydrated samples. The heat capacity of the H2O-hydrated CA(1-7)M(2-9) peptide was measured by adiabatic calorimetry in the temperature range 5-300 K, for different hydration levels. The glass transition and water crystallization temperatures were derived in each case. The existence of different types of water was inferred and their amounts calculated. The heat capacities as obtained from direct calorimetric measurements were compared to the values derived from the neutron spectroscopy by way of integrating appropriately normalized VDOS functions. While there is remarkable agreement with respect to both temperature dependence and glass transition temperatures, the results also show that the VDOS derived part represents only a fraction of the total heat capacity obtained from calorimetry. Finally our results indicate that both hydration water and the peptide are involved in the experimentally observed transitions.

Surface Grafted MSI-78A Antimicrobial Peptide has High Potential for Gastric Infection Management.

As we approach the end of the antibiotic era, newer therapeutic options, such as antimicrobial peptides (AMPs), are in urgent demand. AMP surface grafting onto biomaterials has been described as a good strategy to overcome problems associated with their in vivo stability. Helicobacter pylori is among the bacteria that pose greatest threat to human health, being MSI-78A one of the few bactericidal AMPs against this bacterium. Here, we report that MSI-78A grafted onto model surfaces (Self-Assembled Monolayers -SAMs), in a concentration of 30.3 ± 1.2 ng/cm2 determined by quartz crystal microbalance with dissipation (QCM-D), was able to kill, by contact, 98% of planktonic H. pylori in only 2 h. This fact was not verified against the control bacteria (Staphylococcus epidermidis), although the minimal inhibitory concentration (MIC) of MSI-78A in solution is much lower for S. epidermidis (2 µg/mL) than for H. pylori (64 µg/mL). Our results also demonstrated that, in opposite to other bacteria, H. pylori cells were attracted to ethylene glycol terminated (antiadhesive) surfaces, which can explain the high bactericidal potential of grafted MSI-78A. This proof of concept study establishes the foundations for development of MSI-78A grafted nanoparticles for gastric infection management within a targeted nanomedicine concept.

Dhvar5 antimicrobial peptide (AMP) chemoselective covalent immobilization results on higher antiadherence effect than simple physical adsorption.

Bacterial colonization and subsequent biofilm formation is still one of the major problems associated with medical devices. Antimicrobial peptides (AMP) immobilization onto biomaterials surface is a promising strategy to avoid bacterial colonization. However, a correct peptide orientation and exposure from the surface is essential to maintain AMP antimicrobial activity. This work aims to evaluate the effect of the immobilization on antibacterial activity of Dhvar5 (LLLFLLKKRKKRKY), an AMP with a head-to-tail amphipathicity. Dhvar5 was linked to thin chitosan coatings in i) a controlled orientation and exposure, testing covalent immobilization of its N- or C-terminus and using spacers with different lengths and flexibilities or in ii) a random orientation by physical adsorption. Chitosan coating was chosen due to its antimicrobial properties and readiness to be functionalized. Surface characterization demonstrated the chemoselective immobilization of the peptide with different spacers in a similar concentration (∼2 ng/cm2). Efficacy assays demonstrated that covalent immobilization of Dhvar5 exposing its cationic end, improves the chitosan coating antimicrobial effect by decreasing Methicillin-resistant Staphylococcus aureus (MRSA) colonization. This effect was enhanced when longer spacers were used independently of their flexibility. In opposite, immobilized Dhvar5 exposing its hydrophobic end has no effect on bacterial adhesion to chitosan, and when adsorbed in a random orientation even induces bacterial adhesion to chitosan coating.

Characterization of hLF1-11 immobilization onto chitosan ultrathin films, and its effects on antimicrobial activity.

hLF1-11 (GRRRRSVQWCA) is an antimicrobial peptide (AMP) with high activity against methicillin-resistant Staphylococcus aureus (MRSA), the most prevalent species in implant-associated infection. In this work, the effect of the surface immobilization on hLF1-11 antimicrobial activity was studied. Immobilization was performed onto chitosan thin films as a model for an implant coating due to its reported osteogenic and antibacterial properties. Chitosan thin films were produced by spin-coating on gold surfaces. hLF1-11 was immobilized onto these films by its C-terminal cysteine in an orientation that exposes the antimicrobial activity-related arginine-rich portion of the peptide. Two levels of exposure (with and without a polyethylene glycol (PEG) spacer) were analyzed. Covalent immobilization was further compared with the AMP physical adsorption onto chitosan films. Surfaces were characterized using ellipsometry, contact angle measurements, atomic force microscopy, infrared and X-ray photoelectron spectroscopies and using a fluorimetric assay for hLF1-11 quantification. Surface antimicrobial activity was assessed through surface adhesion and viability assays using an MRSA (S. aureus ATCC 33591). The incorporation of hLF1-11 increased significantly bacterial adhesion to chitosan films. However, the presence of hLF1-11, namely when immobilized through a PEG spacer, decreased the viability of adherent bacteria with regard to the control surface. These results demonstrated that hLF1-11 after covalent immobilization by its cysteine can maintain activity, particularly if a spacer is applied. However, further studies, exploring the opposite orientation or the same C-terminal orientation, but non-cysteine related, can help to clarify the potential of the hLF1-11 immobilization strategy.

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide.

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices.

Effect of surface coating on the biodistribution profile of gold nanoparticles in the rat.

Successful application of gold nanoparticles (AuNPs) in biomedicine requires extensive safety assessment for which biokinetic studies are crucial. We evaluated the biodistribution of AuNPs (∼20 nm) with different surface coatings: citrate, 11-MUA and 3 pentapeptides, CALNN, CALND and CALNS, after i.v. administration to rats (0.6-1 mg Au/kg). Biodistribution was evaluated based on Au tissue content measured by GFAAS. Citrate-AuNPs were rapidly removed from circulation with 60% of the injected dose depositing in the liver. Thirty minutes post-injection, the lungs presented about 6% of the injected dose with levels decreasing to 0.7% at 24 h. Gold levels in the spleen were of 2.6%. After 24 h, liver presented the highest Au level, followed by spleen and blood. A similar biodistribution profile was observed for MUA-coated AuNPs compared to Cit-AuNPs at 24h post-injection, while significantly higher levels of peptide-capped AuNPs were found in the liver (74-86%) accompanied by a corresponding decrease in blood levels. TEM analysis of liver slices showed AuNPs in Kupffer cells and hepatocytes, trapped inside endosomes. Our data demonstrate that AuNPs are rapidly distributed and that the liver is the preferential accumulation organ. Peptide capping significantly increased hepatic uptake, showing the influence of AuNPs functionalization in biodistribution.