Review of a viral peptide nanosystem for intracellular delivery

The internalization of bioactive molecules is one of the most critical problems to overcome in theranostics. In order to improve pharmacokinetic and pharmacodynamic proper- ties, synthetic transporters are widely investigated. A new nanotechnological transporter, gH625, is based on a viral peptide sequence derived from the herpes simplex virus type 1 glycoprotein H (gH) that has proved to be a useful delivery vehicle, due to its intrinsic properties of inducing membrane perturbation. The peptide functionalization with several kinds of nanoparticles like quantum dots, dendrimers, and liposomes could be of particular interest in biomedical applica- tions to improve drug release within cells, to increase site-specific action, and eventually to reduce related cytotoxicity. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. (DOI: 10.1117/1.JNP.7.071599

Septic Shock by Gram-Negative Infections: Role of Outer Membrane Proteins

The magnitude of septic shock as a clinical problem is often understated. Despite advances in our ability to diagnose and treat infectious diseases, severe sepsis leading to shock due to gram-negative infections remains one of the leading causes of mortality worldwide. Septic shock develops because of a disregulation in the host response, and the mechanisms initially recruited to fight infection produce life-threatening tissue damage and death. Recent research has witnessed a significant increase in our understanding of host-pathogen interactions, particularly in the area of innate immunity and the molecular recognition of gram-positive and gram-negative bacteria. Important new mediators of sepsis and novel mechanisms of host-cell toxicity have been identified and, together with clinical trials targeting pathways considered central to sepsis pathogenesis, provide new insight into the molecular and cellular basis of sepsis for the formulation of new strategies of intervention. Research on septic shock pathogenesis by gram-negative bacteria is mainly focused on the understanding of the molecular and cellular role played by lipopolysaccharide (LPS). Strong experimental evidence and clinical observations suggest that the release of proinflammatory cytokine mediators by LPS-responsive cells (mainly macrophages, endothelial cells and neutrophils) in response to toxic products sets in motion the genetic and physiologic program that manifests as shock. The best characterized of these toxic components is LPS, which is considered as a paradigm for other less well-characterized toxic microbial molecules. The immune protection stimulated by highly purified LPS in animals does not resolve the symptomatology of septic shock, while LPS mixed to outer membrane proteins shows a better protective activity. Several studies evidence the major role played by outer membrane proteins in the molecular interaction between the host cell and the gram-negative bacteria. Endotoxin-associated proteins consist of a complex of several major proteins that are intimately associated with the LPS. Very little is known about release of non-LPS gramnegative outer membrane components such as OMPs in sepsis. Among the OMPs, porins have been shown to play an important role in pathogenesis of bacterial infections. Porins were pyrogenic in rabbits and elicited a localized reaction when used as the sensiting and eliciting agent. Porins were also shown to kill D-galactosamine sensitized LPS-responsive and LPS-unresponsive mice. Treatment of Human Umbilical Vein Endothelial Cells: (HUVEC) with porins increased the transmigration of different leukocyte populations, inparticular of neutrophils. Porins by several gram-negative bacteria induce cytokine release by human leukocytes as well as enhancement of cytokine gene expression. Also, other components of the bacterial envelope are important in the induction and pathogenesis of septic shock such as bacterial lipoproteins (LP). As anti-LPS therapies does not seem to improve by the addition of proteins from the outer membrane or small fragments of these proteins, a great alternative to existing strategies will involve the blockage of signal transduction pathways, cytokine and inflammatory mechanisms

Fusogenic domains in herpes simplex virus type 1 glycoprotein H.

Infection of eukaryotic cells by enveloped viruses requires fusion between the viral envelope and the cellular plasma or endosomal membrane. The actual merging of the two membranes is mediated by viral envelope glycoproteins, which generally contain a highly hydrophobic region termed the fusion peptide. The entry of herpesviruses is mediated by three conserved proteins: glycoproteins B, H (gH), and L. However, how fusion is executed remains unknown. Herpes simplex virus type 1 gH exhibits features typical of viral fusion glycoproteins, and its ectodomain seems to contain a putative internal fusion peptide. Here, we have identified additional internal segments able to interact with membranes and to induce membrane fusion of large unilamellar vesicles. We have applied the hydrophobicity-at-interface scale proposed by Wimley and White (Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848) to identify six hydrophobic stretches within gH with a tendency to partition into the membrane interface, and four of them were able to induce membrane fusion. Experiments in which equimolar mixtures of gH peptides were used indicated that different fusogenic regions may act in a synergistic way. The functional and structural characterization of these segments suggests that herpes simplex virus type 1 gH possesses several fusogenic internal peptides that could participate in the actual fusion event

Viral fusion peptides induce several signal transduction pathway activations that are essential for interleukin-10 and beta-interferon production

Objectives: The deciphering of intracellular signaling pathways that are activated by the interaction between viral fusion peptides and cellular membranes are important for the understanding of both viral replication strategies and host defense mechanisms. Methods: Fusion peptides of several enveloped viruses belonging to different virus families were prepared by standard 9-fluorenylmethoxycarbonyl polyamine solid-phase synthesis and used to stimulate U937 cells in vitro to analyze the phosphorylation patterns of the signaling pathways (PKC, Src, Akt, and MAPK pathways). Immunoprecipitation and Western blotting were carried out by using phosphospecific antibodies. All samples were also assayed for the presence of IL-10 and IFN-beta by ELISA and activation of nuclear factors (AP-1 and NF-kappa B). Results: We have demonstrated that hydrophobic domains of fusion proteins are able to induce several transduction pathways that lead to cytokine (IFN-beta and IL-10) production, an event that appears to be dependent on early activation of AP-1 and NF-kappa B. Conclusions: The results obtained on the signaling activity of fusion peptides from different viruses enabled us to shed some light on the complex mechanism of viral entry and more precisely we focused on the exact signaling event induced by hydrophobic domains characteristic of fusion peptides interacting with the cell membrane. Copyright (C) 2010 S. Karger AG, Base

Quantitative and qualitative effect of gH625 on the nanoliposome-mediated delivery of mitoxantrone anticancer drug to HeLa cells

The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin to the cytoplasm of cancer cells, while the mechanism of this enhancement has not yet been understood. Here, in order to get a better insight into the role of gH625 on the mechanism of liposome-mediated drug delivery, we treated HeLa cells with liposomes functionalized with gH625 and loaded with MTX; liposome were characterized in terms of their physico-chemical properties and drug release kinetics. To quantify the MTX uptake and to study the subcellular drug distribution and interaction, we took advantage of the intrinsic fluorescence of MTX and of the fluorescence-based techniques like fluorescence-activated cell sorting (FACS) and confocal spectral imaging (CSI). gH625 liposomes showed an enhanced staining of the internalized drug is observed mainly in hydrophobic regions of the cytoplasm, where the increased presence of an oxidative metabolite of the drug is observed. MTX delivery with gH625-decorated nanoliposomes enhances the quantity of both the intracellular drug and of its oxidative metabolite and contributes to higher anticancer efficacy of the drug

Competitiveness during Dual-Species Biofilm Formation of Fusarium oxysporum and Candida albicans and a Novel Treatment Strategy

During an infection, a single or multispecies biofilm can develop. Infections caused by non-dermatophyte molds, such as Fusarium spp. and yeasts, such as Candida spp., are particularly difficult to treat due to the formation of a mixed biofilm of the two species. Fusarium oxysporum is responsible for approximately 20% of human fusariosis, while Candida albicans is responsible for superficial mucosal and dermal infections and for disseminated bloodstream infections with a mortality rate above 40%. This study aims to investigate the interactions between C. albicans and F. oxysporum dual-species biofilm, considering variable formation conditions. Further, the ability of the WMR peptide, a modified version of myxinidin, to eradicate the mixed biofilm when used alone or in combination with fluconazole (FLC) was tested, and the efficacy of the combination of WMR and FLC at low doses was assessed, as well as its effect on the expression of some biofilm-related adhesin and hyphal regulatory genes. Finally, in order to confirm our findings in vivo and explore the synergistic effect of the two drugs, we utilized the Galleria mellonella infection model. We concluded that C. albicans negatively affects F. oxysporum growth in mixed biofilms. Combinatorial treatment by WMR and FLC significantly reduced the biomass and viability of both species in mature mixed biofilms, and these effects coincided with the reduced expression of biofilm-related genes in both fungi. Our results were confirmed in vivo since the synergistic antifungal activity of WMR and FLC increased the survival of infected larvae and reduced tissue invasion. These findings highlight the importance of drug combinations as an alternative treatment for C. albicans and F. oxysporum mixed biofilms

Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood-brain barrier in rats.

Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood-brain barrier (BBB). In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human disease

Activity of Free and Liposome-Encapsulated Essential Oil from Lavandula angustifolia against Persister-Derived Biofilm of Candida auris

The high virulence of Candida auris, a pathogen fungus considered as a global threat for public health, is due to its peculiar traits such as its intrinsic resistance to conventional antifungals. Its biofilm lifestyle certainly promotes the prolonged survival of C. auris after disinfection or antifungal treatments. In this work, for the first time, we detected persister cells in a biofilm of C. auris in a microwell plate model, following caspofungin treatment. Furthermore, we showed how persisters can progressively develop a new biofilm in situ, mimicking the re-colonization of a surface which may be responsible for recalcitrant infections. Plant-derived compounds, such as essential oils, may represent a valid alternative to combat fungal infections. Here, Lavandula angustifolia essential oil, as free or encapsulated in liposomes, was used to eradicate primary and persister-derived biofilms of C. auris, confirming the great potential of alternative compounds against emergent fungal pathogens. As in other Candida species, the action of essential oils against C. auris involves ROS production and affects the expression of some biofilm-related genes

Antifungal and Antibiofilm Activity of Cyclic Temporin L Peptide Analogues against Albicans and Non-Albicans Candida Species

Temporins are one of the largest families of antimicrobial peptides with both anti-inflammatory and antimicrobial activity. Herein, for a panel of cyclic temporin L isoform analogues, the antifungal and antibiofilm activities were determined against representative Candida strains, including C. albicans, C. glabrata, C. auris, C. parapsilosis and C. tropicalis. The outcomes indicated a significant anti-candida activity against planktonic and biofilm growth for four peptides (3, 7, 15 and 16). The absence of toxicity up to high concentrations and survival after infection were assessed in vivo by using Galleria mellonella larvae, and the correlation between conformation and cytotoxicity was investigated by fluorescence assays and circular dichroism (CD). By combining fluorescence spectroscopy, CD, dynamic light scattering, confocal and atomic force microscopy, the mode of action of four analogues was hypothesized. The results pinpointed that peptide 3 emerged as a non-toxic compound showing a potent antibiofilm activity and represents a promising compound for biomedical applications