Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB)

BACKGROUND:Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. METHODOLOGY/PRINCIPAL FINDINGS:Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. CONCLUSIONS:Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators

Entwicklung eines Bakterien-responsiven Antibiotikumsfreisetzungssystems

A major problem regarding public health is the emergence of antibiotic resistant bacterial strains, especially methicillin resistant Staphylococcus aureus (MRSA). This is mainly attributed to the unnecessary overuse of antimicrobial drugs by patients; however, one aspect that is often neglected is their untargeted mechanism of action, affecting not only the infection itself but also commensal bacteria which are often opportunistic pathogens causing many diseases as well. Therefore, our goal was to develop a bioresponsive antibiotic delivery system triggered by virulence factors. The designed system is comprised of a polymer to enhance its pharmacokinetic profile, a peptide cleavable linker, and the antibiotic agent itself. The bacterial protease aureolysin which is expressed by S. aureus during infections would cleave the linker and partially release the antibiotic which would be still attached to a remaining tetrapeptide. These would be cleaved by a group of proteases naturally present in plasma called aminopeptidases, finally releasing the compound. In the first part of this project, we searched for a suitable sequence to serve as a cleavable linker. It should be sensitive towards the target bacterial protease but not be cleaved by any human enzymes to guarantee the specificity of the system. Therefore, we synthesized three peptide sequences via Solid Phase Peptide Synthesis and incubated them with aureolysin as well as with many human matrix Metalloproteases. The analysis and quantification of enzymatic activity was monitored chromatographically (RP-HPLC). The plasminogen originated sequence was chosen since it was not sensitive towards MMPs, but cleaved by aureolysin. In the second part, we tried to incorporate the chosen peptide sequences as crosslinkers in hydrogel formulations. The purpose was to physically incorporate the antibiotic within the hydrogel, which would be released by the cleavage of those sequences and the consequent loosening the hydrogel net. For that purpose we used a commercially available hydrogel kit with a PVA matrix modified with maleimide, which allows a conjugation reaction with thiol functionalized crosslinkers. Three fluorophores were chosen to serve as antibiotic models and a diffusion assay was performed. Only the glomerular structured Green Fluorescent Protein (GFP) presented a low diffusion rate, thus the aureolysin release assays were performed only using this prototype. Assays showed that with a low hydrogel polymer concentration, the fluorophore either quickly diffused into the medium or was not released at all. The physical incorporation of the antibiotic within the hydrogel pores was therefore abolished as a suitable release approach. For a second attempt, we covalently bound a fluorophore to the linker, which was conjugated to the hydrogel matrix. The incubation with aureolysin and subsequent RP-HPLC analysis showed a peak with the same retention time correspondent to the fragment product after cleavage of the free linker. This is a proof that the concept of linking the peptide sequence to the antibiotic is a promising strategy for its bioresponsive release. Within the third part of this study, we analyzed the degradation of the resulted fragment after aureolysin activity and subsequent full release of the antibiotic by human aminopeptidases. We determined the concentration of those enzymes in human plasma and synthesized the fragment by conjugating the tetrapeptide sequence to aminofluorescein via EDC/NHS reaction. By incubating the construct with the lowest aminopeptidase concentration measured in plasma, the fluorophore was completely released within two hours, showing the efficacy of these enzymes as bioresponsive agents. The last part was the construction of the PEGylated linker-antibiotic. For this purpose we chose the tetracycline like antibiotic chelocardin (CHD) as our prototype. The conjugation of the linker- CHD to the polymer was performed by copper free click chemistry. The cleavage rate of the linker by aureolysin was very similar to the one obtained for the free peptide, indicating that the PEGylation does not interfere on the enzymatic activity. However, by trying to increase the loading ratio of chelocardin onto the polymer, we observed a very low cleavage rate for the system, indicating the formation of aggregates by those constructs. The designed system has proved to be a smart strategy for the delivery on demand of antibiotics in which the drug is only released by the presence of S. aureus during their virulent state.Ein weltweites Problem des Gesundheitswesens ist die Entstehung von antibiotikaresistenten Bakterienstämmen, besonders Methicillin-resistenter Staphylococcus aureus (MRSA). Eine wichtige Ursache für Resistenzentwicklungen ist die unüberlegte Verschreibung von Antibiotika; allerdings das breite Wirkspektrum der meisten Substanzen ist ein stets vernachlässigter Aspekt. Dies betrifft nicht nur die Pathogene selbst, sondern auch die bakterielle Mikroflora des Patienten, die opportunistische Pathogene darstellen und in machen Fallen ebenfalls verschiedene Erkrankungen hervorrufen können. Unser Ziel ist die Entwicklung eines bioresponsiven Freisetzungssystems für Antibiotika. Das System besteht aus einem Polymer zur Optimierung der Pharmakokinetik, einem Peptidlinker sowie dem eigentlichen Antibiotikum. Die bakterielle Protease Aureolysin wird von S. aureus exprimiert, sobald sich das Bakterium in seinem virulenten Zustand befindet. Das Enzym schneidet den Linker, wodurch das Antibiotikum zum Teil freigesetzt wird. Da es noch an Aminosäureartefakte gebunden ist, muss es im Anschluss durch eine Aminopeptidase, einer Gruppe von Exoproteasen des humanen Plasmas, abgespalten werden. Die erste Phase des Projektes war die Suche nach einer passenden Peptidsequenz, die als Linker geeignet ist. Diese soll nur durch die Zielprotease und nicht durch andere humane Proteasen geschnitten werden, um die Spezifizität des Systems zu gewährleisten. Es wurden drei Sequenzen ausgewählt und mittels Festphasen-Peptidsynthese hergestellt. Diese wurden mit Aureolysin sowie humanen Matrix-Metalloproteasen (MMP) inkubiert; die Produkte wurden chromatographisch (RP-HPLC) charakterisiert und die enzymatische Aktivität bestimmt. Die von Plasminogen abgeleitete Sequenz wurde von keiner der Matrix-Metalloproteasen geschnitten, wohl aber von Aureolysin. Eine ausführliche Analyse des Aureolysin-Verdaus zeigte, dass der Linker innerhalb weniger Stunden komplett geschnitten wird. In der zweiten Phase wurde die Peptidsequenz als Crosslinker in verschiedene Hydrogelmatrices inkorporiert. Die Strategie war der physikalische Einschluss des Antibiotikums in das Hydrogel und die anschließende Freisetzung durch Spaltung dieser Sequenzen und Lockerung des Hydrogelnetzes auf molekularer Ebene. Hierfür wurde ein kommerzielles Hydrogelkit mit Maleinsäureamid-modifizierter PVA Matrix verwendet, die mit Thiol-funktionalisierten Linkern konjugiert werden können. Drei verschiedene Fluorophore wurden als Modelle für die Diffusionsversuche verwendet. Nur das glomeruläre green fluorescent protein (GFP) besaß eine ausreichend niedrige Diffusionskonstante und wurde deshalb als Prototyp für die weiteren Schneidversuche verwendet. Die Ergebnisse zeigen, dass der Fluorophor bei niedrigen Matrixkonzentrationen schnell aus den Poren in das umgebende Medium diffundiert, während er bei höheren Konzentrationen nicht freigesetzt wird. Die physikalische Inkorporierung des Antibiotikums wurde aus diesen Gründen verworfen und nicht durchgeführt. Als zweiter Versuch wurde der Fluorophor kovalent an den Linker gekoppelt, welcher im Anschluß an die Matrix konjugiert wurde. Die Inkubation mit Aureolysin und die nachfolgende RP-HPLC-Analyse zeigte einen Peak bei der Retentionszeit entsprechend dem Fragmentprodukt, das durch Inkubation des freien Linkers entsteht. Die kovalente Bindung zwischen der antimikrobiellen Substanz und dem Linker ist eine vielversprechende Strategie für eine bio-responsive Freisetzung. In der dritten Phase des Projektes wurde die Zersetzung des resultierenden Fragments nach Aureolysin-Verdau und die anschließende vollständige Freisetzung des Antibiotikums durch humane Aminopeptidasen untersucht. Die Konzentration an Aminopeptidasen im humanen Plasma wurde bestimmt und die durch Aureolysin entstehende Peptidsequenz an Aminofluorescein mittels EDC/NHS-Reaktion gekoppelt. Die Inkubation des Konstruktes mit der niedrigsten Aminopeptidase-Konzentration, die im Plasma bestimmt werden konnte zeigte, dass der Fluorophor in zwei Stunden vollständig freigesetzt wurde. Die letzte Phase hat sich mit der PEGylierung des Linker-Antibiotikum-Komplexes beschäftigt. Das Tetracyclin-analoge Antibiotikum Chelocardin wurde als Prototyp ausgewählt und am Helmholtz-Institut für Pharmazeutische Forschung des Saarlandes synthetisiert. Die Konjugation des Linker-CHD-Konstruktes an das Polymer wurde mittels kupferfreier Click-Chemie durchgeführt. Der PEGylierte Linker wurde in einer ähnlichen Rate durch Aureolysin geschnitten wie der freie Linker, was beweist, dass das Polymer keinen Einfluss auf die enzymatische Aktivität hat. Allerdings wurde während der Optimierung der Beladung von CHD je Polymermolekül eine sehr niedrige Freisetzung des Antibiotikums beobachtet, was durch Aggregatbildung der Konstrukte erklärt werden kann. Das entwickelte System ist eine interessante Delivery-Strategie für Antibiotika, welche hierdurch nur durch virulente S. aureus-Erreger freigesetzt werden

Analysis of the effects of mesoporous silica particles SBA-15 and SBA-16 in streptococcus pneumoniae transformation process

Streptococcus pneumoniae are natural competent bacteria which requires the presence of a pheromone-like molecule to do the transformation process. This study verified the influence of mesoporous silica (SBA-15 and SBA-16) on the transformation process in S. pneumoniae using a donor DNA obtained from a mutant strain of this microorganism (Sp360luxS). The results showed that mesoporous silica SBA-15 and SBA-16 particles doubled the transformation ratio frequency compared with negative control (without nanoparticles) in using SBA-15 (ratio 1.81 +/- 0.04) and SBA-16 (ratio 2.18 +/- 0.22). We demonstrated the how mesoporous silica nanoparticles were able to increase the pneumococcus transformations, which could possibly lead to the acquisition of virulence factor genes and resistance of antibiotics641127132CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloSem informação307494/2016-02011/14079-2; 2013/22127-2; 2014/50906-

Effects of PHMB on <i>L</i>. <i>major</i> morphology and behavior.

<p>(b and d) TEM images of <i>L</i>. <i>major</i> promastigotes (right) showing morphological changes such as shrinkage, extensive cytoplasmic vacuolization (V), marked loss of cytosolic contents and condensed nucleus (N). Images b and d were taken after treatment with 2 μM PHMB for 48 and 24 h, respectively. (a and c) The untreated controls (left) show normal elongated morphology of promastigotes with intact and clear distinct kinetoplast (kDNA), N, mitochondrion (M), lipid vacuoles (LV) and glycosome (G). (e-h) Light microscopy images showing morphological and behavioral changes (no more clamp formation as indicated by arrowheads) after treatment with 2 μM PHMB for 24 h. Scale bars = (a) 1.1 μm, (b) 0.6 μm, (c and d) 0.25 μm, (e and f) 7.5 μm and (g and h) 25 μm.</p

Effects of CpG ODN and PHMB/CpG ODN polyplexes on cytokine production by BMDM.

<p>The bar graphs show the levels of (a-b) IL-6, (c-d) IL-10 and (e-f) IL-12 production after CpG ODN or PHMB/CpG ODN polyplexes were added to BMDM that had been pre-activated with CpG ODN (15 μg/ml) for 30 min and further incubated for 48 h. Controls were cells without any pre-stimulation and the 0 μM doses were cells pre-stimulated by CpG ODN but without any further stimulation by CpG ODN or the complex. The concentrations shown for the polyplexes represent the dose of PHMB and the concentrations of CpG ODN are half of the indicated doses. The error bars show standard errors from three independent experiments.</p

Antileishmanial and cytotoxic effects of PHMB and PHMB/DNA polyplexes.

<p>The tables show antileishmanial efficacy, cytotoxicity and selectivity of the compounds. The IC₅₀ values and/or selectivity index (SI) of the compounds as compared to standard antileishmanial drugs are shown. The table values are the average IC₅₀ values of 3–6 independent experiments for each compound. IC₅₀ = minimum concentration of the substances required to kill 50% of the population; SI = mean IC₅₀ against BMDM /mean IC₅₀ against <i>L</i>. <i>major</i> amastigotes; ND = not determined.</p

Physicochemical characterization of PHMB/CpG ODN nanopolyplexes.

<p>The table shows particle size and zeta potential determination by using DLS and ELS with their PDI. The table summarizes the results of three independent experiments. Size distribution and zeta potential measurements were performed using Millipore water and ISA water (0.15M KCl), respectively.</p

PHMB/DNA interactions and physicochemical characterization of polyplexes.

<p>Formation of PHMB/DNA polyplexes confirmed by 1% agarose gel, TEM and color change. (a) PHMB/gDNA polyplexes, (b) TEM picture showing PHMB/CpG ODN polyplexes formation at 2:1 ratio, (c) PHMB/CpG ODN polyplexes, (d) temporary turbidity change during PHMB/CpG ODN complexation, (e) PHMB/CpG-R polyplexes and (f) the same gel (e) with fluorescence measurement of CpG-R. With the exceptions of negative control and PHMB alone, all wells contained equal amounts (weight) of DNA with various concentrations of PHMB. M represents 2-Log DNA ladder (0.1–10.0 kb, New England Biolabs) and N represents water used as a negative control. All indicated PHMB/DNA ratios are in relative weight (w/w). Indicates two month old PHMB/CpG ODN polyplexes. Scale bar = 300 nm.</p

Cellular uptake of PHMB and CpG ODN, and their polyplxes by BMDM.

<p>The overlay histograms show time dependent uptake of (a) PHMB-FITC and (b) PHMB-FITC/CpG ODN polyplexes into macrophages. Whereas (c) shows uptake of CpG-R by BMDM as polyplex form compared to its free form. The histograms are representative of three independent experiments.</p

Mechanism(s) of action of PHMB on <i>L</i>. <i>major</i> promastigotes.

<p>PHMB disrupted membrane integrity and condensed DNA in <i>L</i>. <i>major</i> promastigotes. Representative FACS histogram (a) propidium iodide (PI) and (b) YO-PRO-1dye staining of promastigotes after treatment with 2 μM PHMB for indicated time points, showing time-dependent effect of PHMB on the membrane integrity. Heat-killed promastigotes and amphotericin B at 2 μM concentration were used as positive controls. (c) Fluorescent microscopy analysis showing condensed and damaged DNA materials of <i>L</i>. <i>major</i> promastigotes after treatment with PHMB at 2 μM for 48 h as compared to the mock control (distilled water). Scale bars = 5 μm.</p