Antimicrobial magnetic nanoparticles based-therapies for controlling infectious diseases.

In the last years, the antimicrobial resistance against antibiotics has become a serious health issue, arise as global threat. This has generated a search for new strategies in the progress of new antimicrobial therapies. In this context, different nanosystems with antimicrobial properties have been studied. Specifically, magnetic nanoparticles seem to be very attractive due to their relatively simple synthesis, intrinsic antimicrobial activity, low toxicity and high versatility. Iron oxide NPs (IONPs) was authorized by the World Health Organization for human used in biomedical applications such as in vivo drug delivery systems, magnetic guided therapy and contrast agent for magnetic resonance imaging have been widely documented. Furthermore, the antimicrobial activity of different magnetic nanoparticles has recently been demonstrated. This review elucidates the recent progress of IONPs in drug delivery systems and focuses on the treatment of infectious diseases and target the possible detrimental biological effects and associated safety issues.

The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5.

Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity.

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae).

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.

Identification of a novel antimicrobial peptide from Brazilian coast coral Phyllogorgia dilatata.

The marine ecosystem is able to provide enormous biomolecule diversity that could be used for treatment of various diseases. In this highly competitive environment, organisms need chemical barriers to reduce or avoid microorganism contamination. Among the molecules that protect these animals the antimicrobial peptides (AMPs) are included. In the present study, crude extracts of coral coral specimens Carijoa riisei, Muriceopsis sulphurea, Neospongodes atlantica, Palythoa caribeorum, Phyllogorgia dilatata and Plexaurella grandiflora were challenged against multiple Grampositive and -negative bacteria showing different activities. P. dilatata crude extract showed the antibacterial activity, and was ammonium-sulfate (0-40%) fractionated, being able to control the growth of K. pneumoniae, S. flexineri and S. aureus. Rich-fraction was further purified by using Amicon® Ultra Centrifugal 10 kDa associated with reversed-phase HPLC chromatography (C18), producing the peptide named Pd-AMP1. Pd-AMP1 was able to inhibit S. aureus development. Mass spectrometry analyses showed a monoisotopic mass of 5372.66 Da and N-terminal sequence showed no significant match with databank. In this view, the prospecting of protein biomolecules and biotechnological potential from marine animals is still little explored and may serve as an alternative to common antibiotics.

LPS inmobilization on porous and non-porous supports as an approach for the isolation of anti-LPS host-defense peptides.

Lipopolysaccharides (LPSs) are the major molecular component of the outer membrane of Gram-negative bacteria. This molecule is recognized as a sign of bacterial infection, responsible for the development of local inflammatory response and, in extreme cases, septic shock. Unfortunately, despite substantial advances in the pathophysiology of sepsis, there is no efficacious therapy against this syndrome yet. As a consequence, septic shock syndrome continues to increase, reaching mortality rates over 50% in some cases. Even though many preclinical studies and clinical trials have been conducted, there is no Food and Drug Administration-approved drug yet that interacts directly against LPS. Cationic host-defense peptides (HDPs) could be an alternative solution since they possess both antimicrobial and antiseptic properties. HDPs are small, positively charged peptides which are evolutionarily conserved components of the innate immune response. In fact, binding to diverse chemotypes of LPS and inhibition of LPS-induced pro-inflammatory cytokines from macrophages have been demonstrated for different HDPs. Curiously, none of them have been isolated by their affinity to LPS. A diversity of supports could be useful for such biological interaction and suitable for isolating HDPs that recognize LPS. This approach could expand the rational search for anti-LPS HDPs.

Host defense peptides: an alternative as antiinfective and immunomodulatory therapeutics.

Host defense peptides are conserved components of innate immune response present among all classes of life. These peptides are potent, broad spectrum antimicrobial agents with potential as novel therapeutic compounds. Also, the ability of host defense peptides to modulate immunity is an emerging therapeutic concept since its selective modulation is a novel antiinfective strategy. Their mechanisms of action and the fundamental differences between pathogens and host cells surfaces mostly lead to a not widely extended microbial resistance and to a lower toxicity toward host cells. Biological libraries and rational design are novel tools for developing such molecules with promising applications as therapeutic drugs.

Screening of antimicrobials from Caribbean sea animals and isolation of bactericidal proteins from the littoral mollusk Cenchritis muricatus.

Marine organisms represent approximately half of the world's biodiversity by virtue of the sea being an immense reservoir of bioactive molecules. Here, antimicrobial crude extract activities of different marine invertebrates from the Caribbean Sea were evaluated. One of the most active, crude extracts was that marine snail Cenchritis muricatus, it was capable of totally inhibiting the development of Staphylococcus aureus and also showed a growth inhibition of 95.9% in Escherichia coli. Aiming to isolate molecules that confirm antimicrobial activity, the crude extract was purified by reversed-phase HPLC C-18 chromatography. Thereafter, one of the obtained fractions preserved this antibacterial activity. Furthermore, SDS-PAGE analysis (15%) showed the presence of two proteins of molecular masses with approximately 10 and 15 kDa, respectively. The first 19 amino acids of both proteins were sequenced by using Edman degradation, yielding unidentified primary structures compared against sequences deposited at NCBI databank. This is the first report of antibacterial proteins isolated from the mollusk Cenchritis muricatus and these proteins could be used as antibiotic alternatives in the aquacultural industry, as well as in agricultural or biomedical research.

Functional characterization of a synthetic hydrophilic antifungal peptide derived from the marine snail Cenchritis muricatus.

Antimicrobial peptides have been found in mollusks and other sea animals. In this report, a crude extract of the marine snail Cenchritis muricatus was evaluated against human pathogens responsible for multiple deleterious effects and diseases. A peptide of 1485.26 Da was purified by reversed-phase HPLC and functionally characterized. This trypsinized peptide was sequenced by MS/MS technology, and a sequence (SRSELIVHQR), named Cm-p1 was recovered, chemically synthesized and functionally characterized. This peptide demonstrated the capacity to prevent the development of yeasts and filamentous fungi. Otherwise, Cm-p1 displayed no toxic effects against mammalian cells. Molecular modeling analyses showed that this peptide possible forms a single hydrophilic α-helix and the probable cationic residue involved in antifungal activity action is proposed. The data reported here demonstrate the importance of sea animals peptide discovery for biotechnological tools development that could be useful in solving human health and agribusiness problems.

Exploring the pharmacological potential of promiscuous host-defense peptides: from natural screenings to biotechnological applications.

In the last few years, the number of bacteria with enhanced resistance to conventional antibiotics has dramatically increased. Most of such bacteria belong to regular microbial flora, becoming a real challenge, especially for immune-depressed patients. Since the treatment is sometimes extremely expensive, and in some circumstances completely inefficient for the most severe cases, researchers are still determined to discover novel compounds. Among them, host-defense peptides (HDPs) have been found as the first natural barrier against microorganisms in nearly all living groups. This molecular class has been gaining attention every day for multiple reasons. For decades, it was believed that these defense peptides had been involved only with the permeation of the lipid bilayer in pathogen membranes, their main target. Currently, it is known that these peptides can bind to numerous targets, as well as lipids including proteins and carbohydrates, from the surface to deep within the cell. Moreover, by using in vivo models, it was shown that HDPs could act both in pathogens and cognate hosts, improving immunological functions as well as acting through multiple pathways to control infections. This review focuses on structural and functional properties of HDP peptides and the additional strategies used to select them. Furthermore, strategies to avoid problems in large-scale manufacture by using molecular and biochemical techniques will also be explored. In summary, this review intends to construct a bridge between academic research and pharmaceutical industry, providing novel insights into the utilization of HDPs against resistant bacterial strains that cause infections in humans.

Optimización de la solución de extracción de moléculas antibacterianas de Cenchritis muricatus (Gastropoda:Littorinidae) / Optimization of extracting solutions of antibacterial molecules from Cenchritis muricatus (Gastropoda:Littorinidae)

Introducción: los moluscos marinos constituyen un reservorio natural de moléculas con potencialidades terapéuticas para el tratamiento de enfermedades infecciosas en momentos en que se han descrito numerosas cepas resistentes a los antibióticos convencionales. Objetivo: comparar 3 soluciones: ácido acético 30 por ciento, metanol 50 por ciento y salina-ácida (NaCl 0,6 mol/L, HCl 1 por ciento) atendiendo a sus capacidades extractivas de moléculas con actividad antibacteriana del molusco marino Cenchritis muricatus. Métodos: para el procesamiento del material biológico se utilizaron las 3 soluciones de extracción y se analizaron los extractos obtenidos de acuerdo con la concentración de proteínas totales y la inhibición del crecimiento bacteriano de cepas de Staphylococcus aureus y Escherichia coli, mediante un bioensayo turbidimétrico en microplacas de 96 pocillos en medio Luria-Bertani. Resultados: se obtuvo mayor concentración de proteínas totales (7,8 mg/mL) con el extracto total de C. muricatus obtenido con la solución salina-ácida. Además con 200 mg/mL de proteínas totales del extracto se obtuvo inhibición significativa (p< 0,001) del crecimiento de S. aureus (12,64 por ciento) y E. coli (12,1 por ciento) respecto al control positivo de inhibición del crecimiento por cloranfenicol. Conclusiones: de acuerdo con los resultados de la comparación entre las soluciones, la solución salina-ácida resultó ser la más eficiente en la extracción de moléculas antibacterianas, probablemente péptidos antimicrobianos de C. muricatus.

Introduction: marine mollusks are natural reservoirs of molecules with therapeutic potential for the treatment of infectious diseases, at a time when many antibiotic-resistant strains are being described. Objective: to compare three solutions: 30 percent acetic acid, 50 percent methanol and saline-acid (NaCl 0.6 mol/L, 1 percent HCl) according to their capacities to extract molecules with antimicrobial activity from the marine mollusk Cenchritis muricatus. Methods: the three extraction solutions were used to process the biological material, and then, the obtained extracts were analyzed in terms of total protein concentration and the bacterial growth inhibition of Staphylococcus aureus and Escherichia coli strains by means of a turbidimetric bioassay using 96 well microplates in Luria-Bertani (LB) culture medium. Results: the highest total protein concentration (7.8 mg/mL) was found in the C. muricatus extract from the saline-acid solution. Additionally, 200 mg/mL of total proteins from the extract caused significant growth inhibition (p<0.001) of S. aureus (12.64 percent) and E. coli (12.1 percent) compared to the positive control of growth inhibition using chloramphenicol. Conclusions: according to these results, the saline-acid solution proved to be more efficient in extracting molecules with antibacterial activity that are likely to be antimicrobial peptides from C. muricatus.

Optimización de la solución de extracción de moléculas antibacterianas de Cenchritis muricatus (Gastropoda: Littorinidae) / Optimization of extracting solutions of antibacterial molecules from Cenchritis muricatus (Gastropoda: Littorinidae)

Introducción: los moluscos marinos constituyen un reservorio natural de moléculas con potencialidades terapéuticas para el tratamiento de enfermedades infecciosas en momentos en que se han descrito numerosas cepas resistentes a los antibióticos convencionales. Objetivo: comparar 3 soluciones: ácido acético 30 por ciento, metanol 50 por ciento y salina-ácida (NaCl 0,6 mol/L, HCl 1 por ciento) atendiendo a sus capacidades extractivas de moléculas con actividad antibacteriana del molusco marino Cenchritis muricatus. Métodos: para el procesamiento del material biológico se utilizaron las 3 soluciones de extracción y se analizaron los extractos obtenidos de acuerdo con la concentración de proteínas totales y la inhibición del crecimiento bacteriano de cepas de Staphylococcus aureus y Escherichia coli, mediante un bioensayo turbidimétrico en microplacas de 96 pocillos en medio Luria-Bertani. Resultados: se obtuvo mayor concentración de proteínas totales (7,8 mg/mL) con el extracto total de C. muricatus obtenido con la solución salina-ácida. Además con 200 mg/mL de proteínas totales del extracto se obtuvo inhibición significativa (p< 0,001) del crecimiento de S. aureus (12,64 por ciento) y E. coli (12,1 por ciento) respecto al control positivo de inhibición del crecimiento por cloranfenicol. Conclusiones: de acuerdo con los resultados de la comparación entre las soluciones, la solución salina-ácida resultó ser la más eficiente en la extracción de moléculas antibacterianas, probablemente péptidos antimicrobianos de C. muricatus(AU)

Introduction: marine mollusks are natural reservoirs of molecules with therapeutic potential for the treatment of infectious diseases, at a time when many antibiotic-resistant strains are being described. Objective: to compare three solutions: 30 percent acetic acid, 50 percent methanol and saline-acid (NaCl 0.6 mol/L, 1 percent HCl) according to their capacities to extract molecules with antimicrobial activity from the marine mollusk Cenchritis muricatus. Methods: the three extraction solutions were used to process the biological material, and then, the obtained extracts were analyzed in terms of total protein concentration and the bacterial growth inhibition of Staphylococcus aureus and Escherichia coli strains by means of a turbidimetric bioassay using 96 well microplates in Luria-Bertani (LB) culture medium. Results: the highest total protein concentration (7.8 mg/mL) was found in the C. muricatus extract from the saline-acid solution. Additionally, 200 mg/mL of total proteins from the extract caused significant growth inhibition (p<0.001) of S. aureus (12.64 percent) and E. coli (12.1 percent) compared to the positive control of growth inhibition using chloramphenicol. Conclusions: according to these results, the saline-acid solution proved to be more efficient in extracting molecules with antibacterial activity that are likely to be antimicrobial peptides from C. muricatus(AU)

[Optimization of extracting solutions of antibacterial molecules from Cenchritis muricatus (Gastropoda: Littorinidae)]. / Optimización de la solución de extracción de moléculas antibacterianas de Cenchritis muricatus (Gastropoda: Littorinidae).

INTRODUCTION: marine mollusks are natural reservoirs of molecules with therapeutic potential for the treatment of infectious diseases, at a time when many antibiotic-resistant strains are being described. OBJECTIVE: to compare three solutions: 30 % acetic acid, 50 % methanol and saline-acid (NaCl 0.6 mol/L, 1 % HCl) according to their capacities to extract molecules with antimicrobial activity from the marine mollusk Cenchritis muricawus. METHODS: the three extraction solutions were used to process the biological material, and then, the obtained extracts were analyzed in terms of total protein concentration and the bacterial growth inhibition of Staphylococcus aureus and Escherichia coli strains by means of a turbidimetric bioassay using 96 well microplates in Luria-Bertani (LB) culture medium. RESULTS: the highest total protein concentration (7.8 microg/mL) was found in the C. muricatus extract from the saline-acid solution. Additionally, 200 microg/mL of total proteins from the extract caused significant growth inhibition (p< 0.001) of S. aureus (12.64 %) and E. coli (12.1 %) compared to the positive control of growth inhibition using chloramphenicol. CONCLUSIONS: according to these results, the saline-acid solution proved to be more efficient in extracting molecules with antibacterial activity that are likely to be antimicrobial peptides from C. muricatus.

Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control.

Antimicrobial peptides are widely expressed in organisms and have been linked to innate and acquired immunities in vertebrates. These compounds are constitutively expressed and rapidly induced at different cellular levels to interact directly with infectious agents and/or modulate immunoreactions involved in defense against pathogenic microorganisms. In invertebrates, antimicrobial peptides represent the major humoral defense system against infection, showing a diverse spectrum of action mechanisms, most of them related to plasma membrane disturbance and lethal alteration of microbial integrity. Marine invertebrates are widespread, extremely diverse, and constantly under an enormous microbial challenge from the ocean environment, itself altered by anthropic influences derived from industrialization and transportation. Consequently, this study reexamines the peptides isolated over the past 2 decades from different origins, bringing phyla not previously reviewed up to date. Moreover, a promising novel use of antimicrobial peptides as effective drugs in human and veterinary medicine could be based on their unusual properties and synergic counterparts as immune response humoral effectors, in addition to their direct microbicidal activity. This has been seen in many other marine proteins that are sufficiently immunogenic to humans, not necessarily in terms of antibody generation but as inflammation promoters and recruitment agents or immune enhancers.