Anaerobic bacteria growth in the presence of cathelicidin LL-37 and selected ceragenins delivered as magnetic nanoparticles cargo.

BACKGROUND: Cationic antibacterial peptides (CAPs) and synthetic molecules mimicking the amphiphilic structure of CAPs, such as ceragenins, are promising compounds for the development of new antimicrobials. RESULTS: We tested the in vitro activity of ceragenins CSA-13 and CSA-131 against several anaerobic bacteria including Bacteroides spp. and Clostridium difficile. We compared results to the activity of cathelicidin LL-37, metronidazole and nanosystems developed by attachment of CSA-13 and CSA-131 to magnetic nanoparticles (MNPs). The antibacterial effect was tested using killing assay and modified CLSI broth microdilution assay. Ceragenins CSA-13 and CSA-131 displayed stronger bactericidal activity than LL-37 or metronidazole against all of the tested bacterial strains. Additionally CSA-131 revealed an enhanced ability to prevent the formation of Bacteroides fragilis and Propionibacterium acnes biofilms. CONCLUSIONS: These data confirmed that ceragenins display antimicrobial activity against a broad range of microorganisms including anaerobic bacteria and deserve further investigations as compounds serving to develop new treatment against anaerobic and mixed infections.

Sphingosine-1-Phosphate Metabolism and Its Role in the Development of Inflammatory Bowel Disease.

Beyond their role as structural molecules, sphingolipids are involved in many important cellular processes including cell proliferation, apoptosis, inflammation, and migration. Altered sphingolipid metabolism is observed in many pathological conditions including gastrointestinal diseases. Inflammatory bowel disease (IBD) represents a state of complex, unpredictable, and destructive inflammation of unknown origin within the gastrointestinal tract. The mechanisms explaining the pathophysiology of IBD involve signal transduction pathways regulating gastro-intestinal system's immunity. Progressive intestinal tissue destruction observed in chronic inflammation may be associated with an increased risk of colon cancer. Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, functions as a cofactor in inflammatory signaling and becomes a target in the treatment of IBD, which might prevent its conversion to cancer. This paper summarizes new findings indicating the impact of (S1P) on IBD development and IBD-associated carcinogenesis.

Recent insights in nanotechnology-based drugs and formulations designed for effective anti-cancer therapy.

The rapid development of nanotechnology provides alternative approaches to overcome several limitations of conventional anti-cancer therapy. Drug targeting using functionalized nanoparticles to advance their transport to the dedicated site, became a new standard in novel anti-cancer methods. In effect, the employment of nanoparticles during design of antineoplastic drugs helps to improve pharmacokinetic properties, with subsequent development of high specific, non-toxic and biocompatible anti-cancer agents. However, the physicochemical and biological diversity of nanomaterials and a broad spectrum of unique features influencing their biological action requires continuous research to assess their activity. Among numerous nanosystems designed to eradicate cancer cells, only a limited number of them entered the clinical trials. It is anticipated that progress in development of nanotechnology-based anti-cancer materials will provide modern, individualized anti-cancer therapies assuring decrease in morbidity and mortality from cancer diseases. In this review we discussed the implication of nanomaterials in design of new drugs for effective antineoplastic therapy and describe a variety of mechanisms and challenges for selective tumor targeting. We emphasized the recent advantages in the field of nanotechnology-based strategies to fight cancer and discussed their part in effective anti-cancer therapy and successful drug delivery.

Magnetic nanoparticles as a drug delivery system that enhance fungicidal activity of polyene antibiotics.

This study was designed to assess the antifungal/anti-biofilm and hemolytic properties of two polyene antibiotics, amphotericin B (AMF) and nystatin (NYS), attached to the surface of magnetic nanoparticles (MNP) against clinical isolates of Candida species and human red blood cells, respectively. The developed nanosystems, MNP@AMF and MNP@NYS, displayed stronger fungicidal activity than unbound AMF or NYS. Synergistic activity was observed with a combination of polyenes and MNPs against all tested Candida strains. Nanosystems were more potent than unbound agents when tested against Candida strains in the presence of pus, and as agents able to prevent Candida biofilm formation. The observed inactivation of catalase Cat1 in Candida cells upon treatment with the nanosystems suggests that disruption of the oxidation-reduction balance is a mechanism leading to inhibition of Candida growth. The significant decrease of polyenes lytic activity against host cells after their attachment to MNPs surface indicates improvement in their biocompatibility.

Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection.

Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13-IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13-IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13-IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds.

Bactericidal activities of cathelicidin LL-37 and select cationic lipids against the hypervirulent Pseudomonas aeruginosa strain LESB58.

Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains.

Polyelectrolyte-mediated increase of biofilm mass formation.

BACKGROUND: Biofilm formation is associated with various aspects of bacterial and fungal infection. This study was designed to assess the impact of diverse natural polyelectrolytes, such as DNA, F-actin, neurofilaments (NFs), vimentin and purified Pf1 bacteriophage on biofilm formation and swarming motility of select pathogens including Pseudomonas aeruginosa associated with lung infections in CF patients. RESULTS: The bacteriophage Pf1 (1 mg/ml) significantly increased biofilm mass produced by Pseudomonas aeruginosa P14, Escherichia coli RS218 and Bacillus subtilis ATCC6051. DNA, F-actin, NFs and Pf1 also increased biofilm mass of the fungal C. albicans 1409 strain. Addition of F-actin, DNA or Pf1 bacteriophage to 0.5% agar plates increased swarming motility of Pseudomonas aeruginosa Xen5. CONCLUSIONS: The presence of polyelectrolytes at infection sites is likely to promote biofilm growth and bacterial swarming.

Bactericidal activity and biocompatibility of ceragenin-coated magnetic nanoparticles.

BACKGROUND: Ceragenins, synthetic mimics of endogenous antibacterial peptides, are promising candidate antimicrobial agents. However, in some settings their strong bactericidal activity is associated with toxicity towards host cells. To modulate ceragenin CSA-13 antibacterial activity and biocompatibility, CSA-13-coated magnetic nanoparticles (MNP-CSA-13) were synthesized. Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to characterize MNP-CSA-13 physicochemical properties. Bactericidal action and ability of these new compounds to prevent Pseudomonas. aeruginosa biofilm formation were assessed using a bacteria killing assay and crystal violet staining, respectively. Release of hemoglobin from human red blood cells was measured to evaluate MNP-CSA-13 hemolytic activity. In addition, we used surface activity measurements to monitor CSA-13 release from the MNP shell. Zeta potentials of P. aeruginosa cells and MNP-CSA-13 were determined to assess the interactions between the bacteria and nanoparticles. Morphology of P. aeruginosa subjected to MNP-CSA-13 treatment was evaluated using atomic force microscopy (AFM) to determine structural changes indicative of bactericidal activity. RESULTS: Our studies revealed that the MNP-CSA-13 nanosystem is stable and may be used as a pH control system to release CSA-13. MNP-CSA-13 exhibits strong antibacterial activity, and the ability to prevent bacteria biofilm formation in different body fluids. Additionally, a significant decrease in CSA-13 hemolytic activity was observed when the molecule was immobilized on the nanoparticle surface. CONCLUSION: Our results demonstrate that CSA-13 retains bactericidal activity when immobilized on a MNP while biocompatibility increases when CSA-13 is covalently attached to the nanoparticle.

Ceragenin CSA-13 as free molecules and attached to magnetic nanoparticle surfaces induce caspase-dependent apoptosis in human breast cancer cells via disruption of cell oxidative balance.

Natural antimicrobial peptides and ceragenins, as non-peptide amphipathic mimics, have been proposed as anti-cancer agents. To date, it has been confirmed that cathelicidin LL-37 and ceragenin CSA-13, both in free form and immobilized on the surface of magnetic nanoparticles (MNP@LL-37, MNP@CSA-13) induce apoptosis in colon cancer cells. Nevertheless, the question remains whether ceragenins, as synthetic analogs of LL-37 peptide and mimicking a number of its properties, act as antineoplastic agents in breast cancer cells, where LL-37 peptide stimulates oncogenesis. Considering potential anticancer activity, we determined whether CSA-13 and MNP@CSA-13 might be effective against breast cancer cells. Our study provides evidence that both CSA-13 and MNP@CSA-13 decreased viability and inhibit proliferation of MCF-7 and MDA-MB-231 cells despite the protumorigenic properties of LL-37 peptide. Flow cytometry-based analyses revealed that ceragenin treatment results in increases in dead and PI-negative/low-viability cells, which was associated with glutathione (GSH) depletion and increased reactive oxygen species (ROS) generation followed by mitochondrial membrane depolarization, caspase activation, and DNA fragmentation. These findings demonstrate that both CSA-13 and MNP@CSA-13 cause disruption of the oxidative balance of cancer cells. This novel mechanism of ceragenin-mediated eradication of cancer cells suggest that these agents may be developed as a possible treatment of breast cancer.

Development of antifungal therapies using nanomaterials.

The number and diversity of chemical structures currently available as antibacterial drugs is much higher compared with the number of active substances in relation to pathogenic fungi. In this review we focus on nanotechnology approaches, which offer promising strategies to create nanoagents that possess broad-spectrum antifungal activity and might overcome mechanisms of antibiotic resistance. Special attention was given to magnetic nanoparticles and their ability to restrict fungal growth directly, which depends on surface chemistry and pathogen strains. We speculate that future developments of new antifungal methods will take advantage of the current knowledge of using of magnetic nanomaterials as anticancer agents based on their ability to induce hyperthermia and enhance photosensitizing processes.

The synthesis and antifungal activity of (20S)-3ß-acetoxy-5α-pregnane-20,16ß-carbolactone against fluconazole - Resistant Candida cells.

An efficient procedure for the synthesis of (20S)-3ß-acetoxy-5α-pregnane-20,16ß-carbolactone is described. Bactericidal and fungicidal activity of the lactone against different bacteria such as MSSA, MRSA, E. coli ESBL, P. aeruginosa and clinical isolates of Candida spp., in planktonic and biofilm growth stage were assessed. Additionally, the affinity of this new compound to microbial plasma membrane and hemoglobin release from human red blood cells were determined using fluorometric and colorimetric assay, respectively. Our studies revealed that the lactone exhibits strong antifungal activity, and the ability to prevent pathogens' biofilm formation. Additionally, upon lactone treatment a significant affinity to fungal, but not to human cell membranes, indicating suitable biocompatibility was observed.

Use of magnetic nanoparticles as a drug delivery system to improve chlorhexidine antimicrobial activity.

Nanotechnology offers new tools for developing therapies to prevent and treat oral infections, particularly biofilm-dependent disorders, such as dental plaques and endodontic and periodontal diseases. Chlorhexidine (CHX) is a well-characterized antiseptic agent used in dentistry with broad spectrum activity. However, its application is limited due to inactivation in body fluid and cytotoxicity toward human cells, particularly at high concentrations. To overcome these limitations, we synthesized nanosystems composed of aminosilane-coated magnetic nanoparticles functionalized with chlorhexidine (MNP@CHX). In the presence of human saliva, MNPs@CHX displayed significantly greater bactericidal and fungicidal activity against planktonic and biofilm-forming microorganisms than free CHX. In addition, CHX attached to MNPs has an increased ability to restrict the growth of mixed-species biofilms compared to free CHX. The observed depolarization of mitochondria in fungal cells treated with MNP@CHX suggests that induction of oxidative stress and oxidation of fungal structures may be a part of the mechanism responsible for pathogen killing. Nanoparticles functionalized by CHX did not affect host cell proliferation or their ability to release the proinflammatory cytokine, IL-8. The use of MNPs as a carrier of CHX has great potential for the development of antiseptic nanosystems.

Sporicidal activity of ceragenin CSA-13 against Bacillus subtilis.

Spore-forming bacteria are a class of microorganisms that possess the ability to survive in extreme environmental conditions. Morphological features of spores assure their resistance to stress factors such as high temperature, radiation, disinfectants, and drying. Consequently, spore elimination in industrial and medical environments is very challenging. Ceragenins are a new class of cationic lipids characterized by a broad spectrum of bactericidal activity resulting from amphipathic nature and membrane-permeabilizing properties. To assess the impact of ceragenin CSA-13 on spores formed by Bacillus subtilis (ATCC 6051), we performed the series of experiments confirming that amphipathic and membrane-permeabilizing properties of CSA-13 are sufficient to disrupt the structure of B. subtilis spores resulting in decreased viability. Raman spectroscopy analysis provided evidence that upon CSA-13 treatment the number of CaDPA-positive spores was clearly diminished. As a consequence, a loss of impermeability of the inner membranes of spores, accompanied by a decrease in spore resistance and killing take place. In addition to their broad antimicrobial spectrum, ceragenins possess great potential for development as new sporicidal agents.

Formulation and candidacidal activity of magnetic nanoparticles coated with cathelicidin LL-37 and ceragenin CSA-13.

Fungal infections caused by Candida spp. represent an emerging problem during treatment of immunocompromised patients and those hospitalized with serious principal diseases. The ever-growing number of fungal strains exhibiting drug resistance necessitates the development of novel antimicrobial therapies including those based on membrane-permeabilizing agents and nanomaterials as drug carriers. In this study, the fungicidal activities of LL-37 peptide, ceragenin CSA-13 and its magnetic derivatives (MNP@LL-37, MNP@CSA-13) against laboratory and clinical strains of C. albicans, C. glabrata and C. tropicalis were evaluated. These experiments confirm the high anti-fungal activity of these well-characterized agents mediated by their interaction with the fungal membrane and demonstrate elevated activity following immobilization of LL-37 and CSA-13 on the surface of magnetic nanoparticles (MNPs). Furthermore, MNP-based nanosystems are resistant to inhibitory factors present in body fluids and effectively inhibit formation of fungal biofilm. Simultaneously, synthesized nanostructures maintain immunomodulatory properties, described previously for free LL-37 peptide and CSA-13 substrate and they do not interfere with the proliferation and viability of osteoblasts, confirming their high biocompatibility.

Utility of blood procalcitonin concentration in the management of cancer patients with infections.

Diagnosis of infections in cancer patients is usually problematic since differentiating between infection and fever of unknown origin is often a considerable clinical challenge. In general, increase concentration of blood procalcitonin (PCT) is associated with severe bacterial infection. PCT with an optimal cutoff level of 0.5 ng/mL seems to be the most helpful biochemical parameter in detecting severe infections, mainly bloodstream infection, in patients with hematological cancers. In all clinical situations, the elevated level of PCT should be carefully analyzed, always with a thorough physical examination and an appropriate microbiological assessment.

Candidacidal Activity of Selected Ceragenins and Human Cathelicidin LL-37 in Experimental Settings Mimicking Infection Sites.

Fungal infections, especially those caused by antibiotic resistant pathogens, have become a serious public health problem due to the growing number of immunocompromised patients, including those subjected to anticancer treatment or suffering from HIV infection. In this study we assessed fungicidal activity of the ceragenins CSA-13, CSA-131 and CSA-192 against four fluconazole-resistant Candida strains. We found that ceragenins activity against planktonic Candida cells was higher than activity of human LL-37 peptide and synthetic cationic peptide omiganan. Compared to LL-37 peptide, ceragenins in the presence of DNase I demonstrated an increased ability to kill DNA-induced Candida biofilm. Microscopy studies show that treatment with LL-37 or ceragenins causes Candida cells to undergo extensive surface changes indicating surface membrane damage. This conclusion was substantiated by observation of rapid incorporation of FITC-labeled CSA-13, CSA-131 or LL-37 peptide into the more lipophilic environment of the Candida membrane. In addition to activity against Candida spp., ceragenins CSA-131 and CSA-192 display strong fungicidal activity against sixteen clinical isolates including Cryptococcus neoformans and Aspergillus fumigatus. These results indicate the potential of ceragenins for future development as new fungicidal agents.

The Role of Cathelicidin LL-37 in Cancer Development.

LL-37 is a C-terminal peptide proteolytically released from 18 kDa human cathelicidin protein (hCAP18). Chronic infections, inflammation, tissue injury and tissue regeneration are all linked with neoplastic growth, and involve LL-37 antibacterial and immunomodulatory functions. Such a link points to the possible involvement of LL-37 peptide in carcinogenesis. An increasing amount of evidence suggests that LL-37 can have two different and contradictory effects--promotion or inhibition of tumor growth. The mechanisms are tissue-specific, complex, and depend mostly on the ability of LL-37 to act as a ligand for different membrane receptors whose expression varies on different cancer cells. Overexpression of LL-37 was found to promote development and progression of ovarian, lung and breast cancers, and to suppress tumorigenesis in colon and gastric cancer. This review explores and summarizes the current views on how LL-37 contributes to immunity, pathophysiology and cell signaling involved in malignant tumor growth.

Core-shell magnetic nanoparticles display synergistic antibacterial effects against Pseudomonas aeruginosa and Staphylococcus aureus when combined with cathelicidin LL-37 or selected ceragenins.

Core-shell magnetic nanoparticles (MNPs) are promising candidates in the development of new treatment methods against infections, including those caused by antibiotic-resistant pathogens. In this study, the bactericidal activity of human antibacterial peptide cathelicidin LL-37, synthetic ceragenins CSA-13 and CSA-131, and classical antibiotics vancomycin and colistin, against methicillin-resistant Staphylococcus aureus Xen 30 and Pseudomonas aeruginosa Xen 5, was assessed alone and in combination with core-shell MNPs. Fractional inhibitory concentration index and fractional bactericidal concentration index were determined by microdilution methods. The potential of combined therapy using nanomaterials and selected antibiotics was confirmed using chemiluminescence measurements. Additionally, the ability of tested agents to prevent bacterial biofilm formation was evaluated using crystal violet staining. In most conditions, synergistic or additive effects were observed when combinations of core-shell MNPs with ceragenins or classical antibiotics were used. Our study revealed that a mixture of membrane-active agents such as LL-37 peptide or ceragenin CSA-13 with MNPs potentialized their antibacterial properties and might be considered as a method of delaying and overcoming bacterial drug resistance.

Growth arrest and rapid capture of select pathogens following magnetic nanoparticle treatment.

Thorough understanding of magnetic nanoparticle (MNP) properties is essential for developing new theranostics. In this study, we provide evidence that non-modified magnetic iron oxide nanoparticles and their functionalized derivatives may be used to restrict growth and capture different pathogens. Coprecipitation of Fe(2+) and Fe(3+) ions in an alkaline solution was used to synthesize MNPs that subsequently were functionalized by gold and aminosilane coating. Transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) were used to assess their physicochemical properties. A significant decrease of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans outgrown from medium after addition of MNPs or their derivatives was observed during 24h culture. Measurement of optical density revealed that using MNPs, these pathogens can be quickly captured and removed (with efficiency reaching almost 100%) from purposely infected saline buffer and body fluids such as human blood plasma, serum, abdominal fluids and cerebrospinal fluids. These effects depend on nanoparticle concentration, surface chemistry, the type of pathogen, as well as the surrounding environment.

Magnetic nanoparticles enhance the anticancer activity of cathelicidin LL-37 peptide against colon cancer cells.

The pleiotropic activity of human cathelicidin LL-37 peptide includes an ability to suppress development of colon cancer cells. We hypothesized that the anticancer activity of LL-37 would improve when attached to the surface of magnetic nanoparticles (MNPs). Using colon cancer culture (DLD-1 cells and HT-29 cells), we evaluated the effects of MNPs, LL-37 peptide, its synthetic analog ceragenin CSA-13, and two novel nanosystems, ie, MNP@LL-37 and MNP@CSA-13, on cancer cell viability and apoptosis. Treatment of cancer cells with the LL-37 peptide linked to MNPs (MNP@LL-37) caused a greater decrease in cell viability and a higher rate of apoptosis compared with treatment using free LL-37 peptide. Additionally, we observed a strong ability of ceragenin CSA-13 and MNP@CSA-13 to induce apoptosis of DLD-1 cells. We found that both nanosystems were successfully internalized by HT-29 cells, and cathelicidin LL-37 and ceragenin CSA-13 might play a key role as novel homing molecules. These results indicate that the previously described anticancer activity of LL-37 peptide against colon cancer cells might be significantly improved using a theranostic approach.