Neutron reflectometry as a powerful tool to elucidate membrane interactions of drug delivery systems.

The last couple of decades have seen an explosion of novel colloidal drug delivery systems, which have been demonstrated to increase drug efficacy, reduce side-effects, and provide various other advantages for both small-molecule and biomacromolecular drugs. The interactions of delivery systems with biomembranes are increasingly recognized to play a key role for efficient eradication of pathogens and cancer cells, as well as for intracellular delivery of protein and nucleic acid drugs. In parallel, there has been a broadening of methodologies for investigating such systems. For example, advanced microscopy, mass-spectroscopic "omic"-techniques, as well as small-angle X-ray and neutron scattering techniques, which only a few years ago were largely restricted to rather specialized areas within basic research, are currently seeing increased interest from researchers within wide application fields. In the present discussion, focus is placed on the use of neutron reflectometry to investigate membrane interactions of colloidal drug delivery systems. Although the technique is still less extensively employed for investigations of drug delivery systems than, e.g., X-ray scattering, such studies may provide key mechanistic information regarding membrane binding, re-modelling, translocation, and permeation, of key importance for efficacy and toxicity of antimicrobial, cancer, and other therapeutics. In the following, examples of this are discussed and gaps/opportunities in the research field identified.

Boosting Membrane Interactions and Antimicrobial Effects of Photocatalytic Titanium Dioxide Nanoparticles by Peptide Coating.

Photocatalytic nanoparticles offer antimicrobial effects under illumination due to the formation of reactive oxygen species (ROS), capable of degrading bacterial membranes. ROS may, however, also degrade human cell membranes and trigger toxicity. Since antimicrobial peptides (AMPs) may display excellent selectivity between human cells and bacteria, these may offer opportunities to effectively "target" nanoparticles to bacterial membranes for increased selectivity. Investigating this, photocatalytic TiO2 nanoparticles (NPs) are coated with the AMP LL-37, and ROS generation is found by C11-BODIPY to be essentially unaffected after AMP coating. Furthermore, peptide-coated TiO2 NPs retain their positive ζ-potential also after 1-2 h of UV illumination, showing peptide degradation to be sufficiently limited to allow peptide-mediated targeting. In line with this, quartz crystal microbalance measurements show peptide coating to promote membrane binding of TiO2 NPs, particularly so for bacteria-like anionic and cholesterol-void membranes. As a result, membrane degradation during illumination is strongly promoted for such membranes, but not so for mammalian-like membranes. The mechanisms of these effects are elucidated by neutron reflectometry. Analogously, LL-37 coating promoted membrane rupture by TiO2 NPs for Gram-negative and Gram-positive bacteria, but not for human monocytes. These findings demonstrate that AMP coating may selectively boost the antimicrobial effects of photocatalytic NPs.

Bioactive Suture with Added Innate Defense Functionality for the Reduction of Bacterial Infection and Inflammation.

Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.

Pulmonary delivery systems for antimicrobial peptides.

Bacterial infections of the respiratory tract cause millions of deaths annually. Several diseases exist wherein (1) bacterial infection is the main cause of disease (e.g., tuberculosis and bacterial pneumonia), (2) bacterial infection is a consequence of disease and worsens the disease prognosis (e.g., cystic fibrosis), and (3) bacteria-triggered inflammation propagates the disease (e.g., chronic obstructive pulmonary disease). Current approaches to combat infections generally include long and aggressive antibiotic treatments, which challenge patient compliance, thereby making relapses common and contributing to the development of antibiotic resistance. Consequently, the proportion of infections that cannot be treated with conventional antibiotics is rapidly increasing, and novel therapies are urgently needed. In this context, antimicrobial peptides (AMPs) have received considerable attention as they may exhibit potent antimicrobial effects against antibiotic-resistant bacterial strains but with modest toxicity. In addition, some AMPs suppress inflammation and provide other host defense functions (motivating the alternative term host defense peptides (HDPs)). However, the delivery of AMPs is complicated because they are large, positively charged, and amphiphilic. As a result of this, AMP delivery systems have recently attracted attention. For airway infections, the currently investigated delivery approaches range from aerosols and dry powders to various self-assembly and nanoparticle carrier systems, as well as their combinations. In this paper, we discuss recent developments in the field, ranging from mechanistic mode-of-action studies to the application of these systems for combating bacterial infections in the airways.

Design and characterization of matrix metalloproteinase-responsive hydrogels for the treatment of inflammatory skin diseases.

Enzyme-responsive hydrogels, formed by step growth photopolymerization of biscysteine peptide linkers with alkene functionalized polyethylene glycol, provide interesting opportunities as biomaterials and drug delivery systems. In this study, we developed stimuli-responsive, specific, and cytocompatible hydrogels for delivery of anti-inflammatory drugs for the treatment of inflammatory skin diseases. We designed peptide linkers with optimized sensitivity towards matrix metalloproteinases, a family of proteolytic enzymes overexpressed in the extracellular matrix of the skin during inflammation. The peptide linkers were crosslinked with branched 4-arm and 8-arm polyethylene glycols by thiol-norbornene photopolymerization, leading to the formation of a hydrogel network, in which the anti-inflammatory Janus kinase inhibitor tofacitinib citrate was incorporated. The hydrogels were extensively characterized by physical properties, in vitro release studies, cytocompatibility with fibroblasts, and anti-inflammatory efficacy testing in both an atopic dermatitis-like keratinocyte assay and an activated T-cell assay. The drug release was studied after single and multiple-time exposure to matrix metalloproteinase 9 to mimic inflammatory flare-ups. Drug release was found to be triggered by matrix metalloproteinase 9 and to depend on type of crosslinker and of the polyethylene glycol polymer, due to differences in architecture and swelling behavior. Moreover, swollen hydrogels showed elastic properties similar to those of extracellular matrix proteins in the dermis. Cell studies revealed limited cytotoxicity when fibroblasts and keratinocytes were exposed to the hydrogels or their enzymatic cleavage products. Taken together, our results suggest multi-arm polyethylene glycol hydrogels as promising matrix metalloproteinase-responsive drug delivery systems, with potential in the treatment of inflammatory skin disease. STATEMENT OF SIGNIFICANCE: Smart responsive drug delivery systems such as matrix metalloproteinase-responsive hydrogels are excellent candidates for the treatment of inflammatory skin diseases including psoriasis. Their release profile can be optimized to correspond to the patient's individual disease state by tuning formulation parameters and disease-related stimuli, providing personalized treatment solutions. However, insufficient cross-linking efficiency, low matrix metalloproteinase sensitivity, and undesirable drug release kinetics remain major challenges in the development of such drug delivery systems. In this study, we address shortcomings of previous work by designing peptide linkers with optimized sensitivity towards matrix metalloproteinases and high cross-linking efficiencies. We further provide a proof-of-concept for the usability of the hydrogels in inflammatory skin conditions by employing a drug release set-up simulating inflammatory flare-ups.

Pulmonary surfactant and drug delivery: Vehiculization of a tryptophan-tagged antimicrobial peptide over the air-liquid interfacial highway.

This work evaluates interaction of pulmonary surfactant (PS) and antimicrobial peptides (AMPs) in order to investigate (i) if PS can be used to transport AMPs, and (ii) to what extent PS interferes with AMP function and vice versa. This, in turn, is motivated by a need to find new strategies to treat bacterial infections in the airways. Low respiratory tract infections (LRTIs) are a leading cause of illness and death worldwide that, together with the problem of multidrug-resistant (MDR) bacteria, bring to light the necessity of developing effective therapies that ensure high bioavailability of the drug at the site of infection and display a potent antimicrobial effect. Here, we propose the combination of AMPs with PS to improve their delivery, exemplified for the hydrophobically end-tagged AMP, GRR10W4 (GRRPRPRPRPWWWW-NH2), with previously demonstrated potent antimicrobial activity against a broad spectrum of bacteria under various conditions. Experiments using model systems emulating the respiratory interface and an operating alveolus, based on surface balances and bubble surfactometry, served to demonstrate that a fluorescently labelled version of GRR10W4 (GRR10W4-F), was able to interact and insert into PS membranes without affecting its biophysical function. Therefore, vehiculization of the peptide along air-liquid interfaces was enabled, even for interfaces previously occupied by surfactants layers. Furthermore, breathing-like compression-expansion dynamics promoted the interfacial release of GRR10W4-F after its delivery, which could further allow the peptide to perform its antimicrobial function. PS/GRR10W4-F formulations displayed greater antimicrobial effects and reduced toxicity on cultured airway epithelial cells compared to that of the peptide alone. Taken together, these results open the door to the development of novel delivery strategies for AMPs in order to increase the bioavailability of these molecules at the infection site via inhaled therapies.

Effect of PEGylation on Host Defense Peptide Complexation with Bacterial Lipopolysaccharide.

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

Membrane Interactions of Virus-like Mesoporous Silica Nanoparticles.

In the present study, we investigated lipid membrane interactions of silica nanoparticles as carriers for the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES). In doing so, smooth mesoporous nanoparticles were compared to virus-like mesoporous nanoparticles, characterized by a "spiky" external surface, as well as to nonporous silica nanoparticles. For this, we employed a combination of neutron reflectometry, ellipsometry, dynamic light scattering, and ζ-potential measurements for studies of bacteria-mimicking bilayers formed by palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol. The results show that nanoparticle topography strongly influences membrane binding and destabilization. We found that virus-like particles are able to destabilize such lipid membranes, whereas the corresponding smooth silica nanoparticles are not. This effect of particle spikes becomes further accentuated after loading of such particles with LL-37. Thus, peptide-loaded virus-like nanoparticles displayed more pronounced membrane disruption than either peptide-loaded smooth nanoparticles or free LL-37. The structural basis of this was clarified by neutron reflectometry, demonstrating that the virus-like nanoparticles induce trans-membrane defects and promote incorporation of LL-37 throughout both bilayer leaflets. The relevance of such effects of particle spikes for bacterial membrane rupture was further demonstrated by confocal microscopy and live/dead assays on Escherichia coli bacteria. Taken together, these findings demonstrate that topography influences the interaction of nanoparticles with bacteria-mimicking lipid bilayers, both in the absence and presence of antimicrobial peptides, as well as with bacteria. The results also identify virus-like mesoporous nanoparticles as being of interest in the design of nanoparticles as delivery systems for antimicrobial peptides.

Structural insights into the combinatorial effects of antimicrobial peptides reveal a role of aromatic-aromatic interactions in antibacterial synergism.

The recent development of plants that overexpress antimicrobial peptides (AMPs) provides opportunities for controlling plant diseases. Because plants employ a broad-spectrum antimicrobial defense, including those based on AMPs, transgenic modification for AMP overexpression represents a potential way to utilize a defense system already present in plants. Herein, using an array of techniques and approaches, we report on VG16KRKP and KYE28, two antimicrobial peptides, which in combination exhibit synergistic antimicrobial effects against plant pathogens and are resistant against plant proteases. Investigating the structural origin of these synergistic antimicrobial effects with NMR spectroscopy of the complex formed between these two peptides and their mutated analogs, we demonstrate the formation of an unusual peptide complex, characterized by the formation of a bulky hydrophobic hub, stabilized by aromatic zippers. Using three-dimensional structure analyses of the complex in bacterial outer and inner membrane components and when bound to lipopolysaccharide (LPS) or bacterial membrane mimics, we found that this structure is key for elevating antimicrobial potency of the peptide combination. We conclude that the synergistic antimicrobial effects of VG16KRKP and KYE28 arise from the formation of a well-defined amphiphilic dimer in the presence of LPS and also in the cytoplasmic bacterial membrane environment. Together, these findings highlight a new application of solution NMR spectroscopy to solve complex structures to study peptide-peptide interactions, and they underscore the importance of structural insights for elucidating the antimicrobial effects of AMP mixtures.

Application of tungsten disulfide quantum dot-conjugated antimicrobial peptides in bio-imaging and antimicrobial therapy.

Two-dimensional (2D) tungsten disulfide (WS2) quantum dots offer numerous promising applications in materials and optoelectronic sciences. Additionally, the catalytic and photoluminescence properties of ultra-small WS2 nanoparticles are of potential interest in biomedical sciences. Addressing the use of WS2 in the context of infection, the present study describes the conjugation of two potent antimicrobial peptides with WS2 quantum dots, as well as the application of the resulting conjugates in antimicrobial therapy and bioimaging. In doing so, we determined the three-dimensional solution structure of the quantum dot-conjugated antimicrobial peptide by a series of high-resolution nuclear magnetic resonance (NMR) techniques, correlating this to the disruption of both model lipid and bacterial membranes, and to several key biological performances, including antimicrobial and anti-biofilm effects, as well as cell toxicity. The results demonstrate that particle conjugation enhances the antimicrobial and anti-biofilm potency of these peptides, effects inferred to be due to multi-dendate interactions for the conjugated peptides. As such, our study provides information on the mode-of-action of such conjugates, laying the foundation for their potential use in treatment and monitoring of infections.

Off-Stoichiometric Thiol-Ene Chemistry to Dendritic Nanogel Therapeutics.

A novel platform of dendritic nanogels is herein presented, capitalizing on the self-assembly of allyl-functional polyesters based on dendritic-linear-dendritic amphiphiles followed by simple cross-linking with complementary monomeric thiols via UV initiated off-stoichiometric thiol-ene chemistry. The facile approach enabled multigram creation of allyl reactive nanogel precursors, in the size range of 190-295 nm, being readily available for further modifications to display a number of core functionalities while maintaining the size distribution and characteristics of the master batch. The nanogels are evaluated as carriers of a spread of chemotherapeutics by customizing the core to accommodate each individual cargo. The resulting nanogels are biocompatible, displaying diffusion controlled release of cargo, maintained therapeutic efficacy, and decreased cargo toxic side effects. Finally, the nanogels are found to successfully deliver pharmaceuticals into a 3D pancreatic spheroids tumor model.

Microfluidics-based self-assembly of peptide-loaded microgels: Effect of three dimensional (3D) printed micromixer design.

In an effort to contribute to research in scalable production systems for polymeric delivery systems loaded with antimicrobial peptides (AMPs), we here investigate effects of hydrodynamic flow conditions on microfluidic particle generation. For this purpose, rapid prototyping using 3D printing was applied to prepare micromixers with three different geometric designs, which were used to prepare Ca2+-cross-linked alginate microgels loaded with the AMP polymyxin B in a continuous process. Based on fluid dynamic simulations, the hydrodynamic flow patterns in the micromixers were designed to be either (i) turbulent with chaotic disruption, (ii) laminar with convective mixing, or (iii) convective with microvortex formation. The physicochemical properties of the microgels prepared with these micromixers were characterized by photon correlation spectroscopy, laser-Doppler micro-electrophoresis, small-angle x-ray scattering, and ellipsometry. The particle size and compactness were found to depend on the micromixer geometry: From such studies, particle size and compactness were found to depend on micromixer geometry, the smallest and most compact particles were obtained by preparation involving microvortex flows, while larger and more diffuse microgels were formed upon laminar mixing. Polymyxin B was found to be localized in the particle interior and to cause particle growth with increasing peptide loading. Ca2+-induced cross-linking of alginate, in turn, results in particle contraction. The peptide encapsulation efficiency was found to be higher than 80% for all investigated micromixer designs; the highest encapsulation efficiency observed for the smallest particles generated by microvortex-mediated self-assembly. Ellipsometry results for surface-immobilized microgels, as well as results on peptide encapsulation, demonstrated electrolyte-induced peptide release. Taken together, these findings demonstrate that rapid prototyping of microfluidics using 3D-printed micromixers offers promises for continuous manufacturing of AMP-loaded microgels. Although the micromixer combining turbulent flow and microvortexes was demonstrated to be the most efficient, all three micromixer designs were found to mediate self-assembly of small microgels displaying efficient peptide encapsulation. This demonstrates the robustness of employing 3D-printed micromixers for microfluidic assembly of AMP-loaded microgels during continuous production.

Peptide-Loaded Microgels as Antimicrobial and Anti-Inflammatory Surface Coatings.

Here we report on covalently immobilized poly(ethyl acrylate- co-methacrylic acid) microgels loaded with the host defense peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), which is derived from human heparin cofactor II, as well as its poly(ethylene glycol)-conjugated (PEGylated) version, KYE28PEG. Peptide loading and release, as well as the consequences of these processes on the microgel and peptide properties, were studied by in situ ellipsometry, confocal microscopy, zeta potential measurements, and circular dichroism spectroscopy. The results show that the microgel-peptide interactions are electrostatically dominated, thus promoted at higher microgel charge density, while PEGylation suppresses peptide binding. PEGylation also enhances the α-helix induction observed for KYE28 upon microgel incorporation. Additionally, peptide release is facilitated at physiological salt concentration, particularly so for KYE28PEG, which illustrates the importance of electrostatic interactions. In vitro studies on Escherichia coli show that the microgel-modified surfaces display potent antifouling properties in both the absence and presence of the incorporated peptide. While contact killing dominates at low ionic strength for the peptide-loaded microgels, released peptides also provide antimicrobial activity in bulk at a high ionic strength. Additionally, KYE28- and KYE28PEG-loaded microgels display anti-inflammatory effects on human monocytes. Taken together, these results not only show that surface-bound microgels offer an interesting approach for local drug delivery of host defense peptides but also illustrate the need to achieve high surface loads of peptides for efficient biological effects.

Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects.

The objective of the present study is the investigation of possibilities for boosting peptide anti-inflammatory effects by tryptophan end-tagging, including identification of underlying mechanisms for this. In doing so, effects of tryptophan end-tagging of KYE21 (KYEITTIHNLFRKLTHRLFRR), a peptide derived from heparin co-factor II, on membrane and lipopolysaccharide (LPS) interactions were investigated by ellipsometry, NMR, fluorescence spectroscopy, and circular dichroism measurements. Through its N-terminal W stretch, WWWKYE21 displays higher membrane binding, liposome rupture, and bacterial killing than unmodified KYE21. Analogously, W-tagging promotes binding to E. coli LPS and to its endotoxic lipid A moiety. Furthermore, WWWKYE21 causes more stable peptide/LPS complexes than KYE21, as evidenced by detailed NMR studies, adopting a pronounced helical conformation, with a large hydrophobic surface at the N-terminus due to the presence of W-residues, and a flexible C-terminus due to presence of several positively charged arginine residues. Mirroring its increased affinity for LPS and lipid A, WWWKYE21 displays strongly increased anti-inflammatory effect due to a combination of direct lipid A binding, peptide-induced charge reversal of cell membranes for LPS scavenging, and peptide-induced fragmentation of LPS aggregates for improved phagocytosis. Importantly, potent anti-inflammatory effects were observed at low cell toxicity, demonstrated for both monocytes and erythrocytes.

Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for Use in Plant Disease Control.

KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYT-LR), the representative sequence of helix D of heparin co-factor II, was demonstrated to be potent against agronomically important Gram-negative plant pathogens Xanthomonas vesicatoria and Xanthomonas oryzae, capable of inhibiting disease symptoms in detached tomato leaves. NMR studies in the presence of lipopolysaccharide provided structural insights into the mechanisms underlying this, notably in relationship to outer membrane permeabilization. The three-dimensional solution structure of KYE28 in LPS is characterized by an N-terminal helical segment, an intermediate loop followed by another short helical stretch, and an extended C terminus. The two termini are in close proximity to each other via aromatic packing interactions, whereas the positively charged residues form an exterior polar shell. To further demonstrate the importance of the aromatic residues for this, a mutant peptide KYE28A, with Ala substitutions at Phe(11), Phe(19), Phe(23), and Tyr(25) was designed, which showed attenuated antimicrobial activity at high salt concentrations, as well as lower membrane disruption and LPS binding abilities compared with KYE28. In contrast to KYE28, KYE28A adopted an extended helical structure in LPS with extended N and C termini. Aromatic packing interactions were completely lost, although hydrophobic interaction between the side chains of hydrophobic residues were still partly retained, imparting an amphipathic character and explaining its residual antimicrobial activity and LPS binding as observed from ellipsometry and isothermal titration calorimetry. We thus present key structural aspects of KYE28, constituting an aromatic zipper, of potential importance for the development of novel plant protection agents and therapeutic agents.

Pronounced peptide selectivity for melanoma through tryptophan end-tagging.

Effects of oligotryptophan end-tagging on the uptake of arginine-rich peptides into melanoma cells was investigated under various conditions and compared to that into non-malignant keratinocytes, fibroblasts, and erythrocytes, also monitoring resulting cell toxicity. In parallel, biophysical studies on peptide binding to, and destabilization of, model lipid membranes provided mechanistic insight into the origin of the selectivity between melanoma and non-malignant cells. Collectively, the results demonstrate that W-tagging represents a powerful way to increase selective peptide internalization in melanoma cells, resulting in toxicity against these, but not against the non-malignant cells. These effects were shown to be due to increased peptide adsorption to the outer membrane in melanoma cells, caused by the presence of anionic lipids such as phosphatidylserine and ganglioside GM1, and to peptide effects on mitochondria membranes and resulting apoptosis. In addition, the possibility of using W-tagged peptides for targeted uptake of nanoparticles/drug carriers in melanoma was demonstrated, as was the possibility to open up the outer membrane of melanoma cells in order to facilitate uptake of low Mw anticancer drugs, here demonstrated for doxorubicin.

Delivery of siRNA Complexed with Palmitoylated α-Peptide/ß-Peptoid Cell-Penetrating Peptidomimetics: Membrane Interaction and Structural Characterization of a Lipid-Based Nanocarrier System.

Proteolytically stable α-peptide/ß-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using fluorophore-labeled analogues. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing in vitro. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of α-peptides/ß-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs.

The Thrombin-Derived Host Defense Peptide GKY25 Inhibits Endotoxin-Induced Responses through Interactions with Lipopolysaccharide and Macrophages/Monocytes.

Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), derived from the C terminus of human thrombin, inhibits proinflammatory responses in vitro and in vivo, but the mode of action is unclear. In this study, we show that GKY25, apart from binding bacterial LPS, also interacts directly with monocytes and macrophages in vitro, ex vivo, and in vivo. Moreover, GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore, GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25, based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages.

Antimicrobial effects of helix D-derived peptides of human antithrombin III.

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense.

Antimicrobial peptides.

With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity.