Insights into the synthesis and mechanism of green synthesized antimicrobial nanoparticles,answer to the multidrug resistance

Emergence of the multidrug resistant human pathogenic strains is posing a serious health challenge. Resistant strains carry mutations which help them to resist conventional drugs. Therefore, it is required to produce more effective agents that are able to degrade the resistant pathogenic bacterial strains. The antimicrobial properties of nanoparticles (NPs) by eco-friendly green synthetic methods have pulled attention everywhere owing to their exceptional properties and small particle size of 100 nm. NPs are considered to belong to a group of antimicrobial agents which have ability to go inside microbial cells and kill them. In this comprehensive review, we are discussing the green synthetic methods used for the synthesis of NPs targeting the microbes. Additionally, several characterization techniques of antimicrobial NPs are also discussed. Subsequently, various methods used for the analysis of antimicrobial activities and their mechanisms are also examined.     

Photodynamic therapy: A special emphasis on nanocarrier-mediated delivery of photosensitizers in antimicrobial therapy

Resistance to antimicrobial drugs is an impending healthcare problem of growing significance. In the post-antibiotic era, there is a huge push to develop new tools for effectively treating bacterial infections. Photodynamic therapy involves the use of a photosensitizer that is activated by the use of light of an appropriate wavelength in the presence of oxygen. This results in the generation of singlet oxygen molecules that can kill the target cells, including cancerous cells and microbial cells. Photodynamic therapy is shown to be effective against parasites, viruses, algae, and bacteria. To achieve high antimicrobial activity, a sufficient concentration of photosensitizer should enter the microbial cells. Generally, photosensitizers tend to aggregate in aqueous environments resulting in the weakening of photochemical activity and lowering their uptake into cells. Nanocarrier systems are shown to be efficient in targeting photosensitizers into microbial cells and improve their therapeutic efficiency by enhancing the internalization of photosensitizers into microbial cells. This review aims to highlight the basic principles of photodynamic therapy with a special emphasis on the use of nanosystems in delivering photosensitizers for improving antimicrobial photodynamic therapy.     

Framework-Reactive Siderophore Analogs as Potential Cell-Selective Drugs. Design and Syntheses of Trimelamol-Based Iron Chelators

Currently, the role of DNA-directed alkylating agents as potential anticancer/ antimicrobial drugs is of wide interest. Most of the alkylating agents used clinically as drugs damage DNA in cells without specificity, and this can lead to undesired toxicity problems. Minimizing serum residence time by targeting the drug to select pathogens or organs might diminish the effects of nonselective reactivity. This paper describes the syntheses and preliminary studies of analogs of siderophores (microbial iron chelators) 2 and 20 that incorporate centers within the siderophore framework capable of generating potent electrophiles (iminium ions), hopefully after directed cellular recognition and uptake. Formation of N-aminals from trimelamol (3) and substituted hydroxamic acid 4 or 5was critical for the design and synthesis of the targets. In preliminary biological testing, compound 2, a trimelamol-based siderophore analog, was active against Escherichia coli X580, illustrating the therapeutic potential of this new type of siderophore-mediated drug design and delivery.     

Antimicrobial Properties and Application of Polysaccharides and Their Derivatives

With the quick emergence of antibiotic resistance and multi-drug resistant microbes, more and more attention has been paid to the development of new antimicrobial agents that have potential to take the challenge. Polysaccharides, as one of the major classes of biopolymers,were explored for their antimicrobial properties and applications, owing to their easy accessibility, biocompatibility and easy modification.Polysaccharides and their derivatives have variable demonstrations and applications as antimicrobial agents and antimicrobial biomaterials. A variety of polysaccharides, such as chitosan, dextran, hyaluronic acid, cellulose, other plant/animal-derived polysaccharides and their derivatives have been explored for antimicrobial applications. We expect that this review can summarize the important progress of this field and inspire new concepts, which will contribute to the development of novel antimicrobial agents in combating antibiotic resistance and drug-resistant antimicrobial infections.     

Identification and design of antimicrobial peptides for therapeutic applications

Indiscriminate use of antibiotics has led to a rapid increase of antibiotic resistance among microbes which has increased the need to develop novel antimicrobial agents to fight various infectious diseases. Peptide antibiotics signify a novel class of therapeutic agents and have been isolated from a wide variety of multi-cellular organisms. Peptide antibiotics have shown broad-spectrum antimicrobial activity and they not only kill different bacteria, but also kill various fungi, parasites, protozoans and cancerous cells. Peptides bear several properties that make them particularly attractive such as their small size, rapid activity and a low chance for development of resistance. Because of these distinct properties, the focus for research on antimicrobial peptides has increased tremendously in the recent years. Despite their potential, only selected cationic antimicrobial peptides have been able to enter in clinical trials. Therefore, there is a pressing need to develop new approaches to identify novel antimicrobial peptide therapeutics replacing conventional antibiotics. Recent findings strongly suggest that one can design a new generation of antimicrobials peptides with a wide range of systemic and topical applications against bacterial infections. In this review, we focus on the identification and design of novel antimicrobial peptides for therapeutic applications based on different approaches and strategies. This review also highlights some recent advances in the study of the molecular basis of anti-microbial activity in these peptides, their current pharmacological and clinical development and future directions and applications.     

Improving the Cellular Selectivity of a Membrane-Disrupting Antimicrobial Agent by Monomer Control and by Taming

Antimicrobial resistance represents a significant world-wide health threat that is looming. To meet this challenge, new classes of antimicrobial agents and the redesign of existing ones will be required. This review summarizes some of the studies that have been carried out in my own laboratories involving membrane-disrupting agents. A major discovery that we made, using a Triton X-100 as a prototypical membrane-disrupting molecule and cholesterol-rich liposomes as model systems, was that membrane disruption can occur by two distinct processes, depending on the state of aggregation of the attacking agent. Specifically, we found that monomers induced leakage, while attack by aggregates resulted in a catastrophic rupture of the membrane. This discovery led us to design of a series of derivatives of the clinically important antifungal agent, Amphotericin B, where we demonstrated the feasibility of separating antifungal from hemolytic activity by decreasing the molecule’s tendency to aggregate, i.e., by controlling its monomer concentration. Using an entirely different approach (i.e., a “taming” strategy), we found that by covalently attaching one or more facial amphiphiles (“floats”) to Amphotericin B, its aggregate forms were much less active in lysing red blood cells while maintaining high antifungal activity. The possibility of applying such “monomer control” and “taming” strategies to other membrane-disrupting antimicrobial agents is briefly discussed.     

Synthesis of Novel Thiazole Derivatives Bearing β-Amino Acid and Aromatic Moieties as Promising Scaffolds for the Development of New Antibacterial and Antifungal Candidates Targeting Multidrug-Resistant Pathogens

Rapidly growing antimicrobial resistance among clinically important bacterial and fungal pathogens accounts for high morbidity and mortality worldwide. Therefore, it is critical to look for new small molecules targeting multidrug-resistant pathogens. Herein, in this paper we report a synthesis, ADME properties, and in vitro antimicrobial activity characterization of novel thiazole derivatives bearing β-amino acid, azole, and aromatic moieties. The in silico ADME characterization revealed that compounds 1–9 meet at least 2 Lipinski drug-like properties while cytotoxicity studies demonstrated low cytotoxicity to Vero cells. Further in vitro antimicrobial activity characterization showed the selective and potent bactericidal activity of 2a–c against Gram-positive pathogens (MIC 1–64 µg/mL) with profound activity against S. aureus (MIC 1–2 µg/mL) harboring genetically defined resistance mechanisms. Furthermore, the compounds 2a–c exhibited antifungal activity against azole resistant A. fumigatus, while only 2b and 5a showed antifungal activity against multidrug resistant yeasts including Candida auris. Collectively, these results demonstrate that thiazole derivatives 2a–c and 5a could be further explored as a promising scaffold for future development of antifungal and antibacterial agents targeting highly resistant pathogenic microorganisms.     

Synthetic mimics of mammalian cell surface receptors: prosthetic molecules that augment living cells

Specific receptors on the surface of mammalian cells actively internalize cell-impermeable ligands by receptor-mediated endocytosis. To mimic these internalizing receptors, my laboratory is studying artificial cell surface receptors that comprise N-alkyl derivatives of 3beta-cholesterylamine linked to motifs that bind cell-impermeable ligands. When added to living mammalian cells, these synthetic receptors insert into cellular plasma membranes, project ligand-binding small molecules or peptides from the cell surface, and enable living cells to internalize targeted proteins and other cell-impermeable compounds. These artificial receptors mimic their natural counterparts by rapidly cycling between plasma membranes and intracellular endosomes, associating with proposed cholesterol and sphingolipid-rich lipid raft membrane microdomains, and delivering ligands to late endosomes/lysosomes. This "synthetic receptor targeting" strategy is briefly reviewed here and contrasted with other related cellular delivery systems. Potential applications of artificial cell surface receptors as molecular probes, agents for cellular targeting, tools for drug delivery, and methods for ligand depletion are discussed. The construction of synthetic receptors as prosthetic molecules, designed to seamlessly augment the molecular machinery of living cells, represents an exciting new frontier in the fields of bioorganic chemistry and chemical biology.     

New strategy for reversing biofilm-associated antibiotic resistance through ferrocene-substituted carborane ruthenium(II)-arene complex

Bacterial biofilms are inherently resistant to antimicrobial agents and are difficult to eradicate with conventional antimicrobial agents, resulting in many persistent and chronic bacterial infections. In this contribution, a new strategy for reversing the biofilm-associated antibiotic resistance has been explored by induction of a carborane ruthenium(II)-arene complex (FcRuSB). Our results demonstrate that the FcRuSB could be utilized as an inducer to efficiently reverse the biofilm-associated antibiotic resistance of multidrug-resistant (MDR) clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa. The induced effect of FcRuSB is correlated with a considerable decrease in the expression of extracellular matrix proteins (EMP) of the two strains. The considerable decrease of the EMP of induced cells, resulting in the reduction of adherence and biofilm formation ability of the two types of MDR pathogens, and then can cause significantly enhanced sensitivity of them to antibiotics.     

Testing antimicrobials against biofilm bacteria

Standard laboratory methods are needed to assess the efficacy of antimicrobial agents that are applied to biofilm bacteria. Existing standard suspension tests and dried surface tests show much greater efficacy than antimicrobial agents applied to biofilms. The greater resistance of biofilm bacteria to antimicrobial agents can be attributed to a number of interacting factors, including reaction and diffusion processes that limit an agent's accessibility to bacteria, phenotypic changes in biofilm bacteria caused by stress, and adaptation of the bacteria. Because biofilm systems are so diverse, a variety of new biofilm tests with features that differ in important ways from existing tests will ultimately be required. For example, the biofilm test apparatus may include a pump and a continuous-flow stirred tank reactor. This report provides an overview of biofilm testing and suggests a strategy for creating standard test methods.     

Bacterial membranes as predictors of antimicrobial potency

A wide range of chemical structures having antimicrobial activity have been studied in an effort to treat the increasing emergence of bacteria that are resistant to traditional antibiotics. These agents have varying degrees of toxicity against different bacterial species. We demonstrate, using members of a novel class of antimicrobial agents, the oligomers of acyllysine, that one cause for the difference in species selectivity is the ability to induce the clustering of anionic lipids, resulting in their segregation into domains. This phenomenon occurs only in bacterial membranes composed of both anionic and zwitterionic lipids and not with bacteria whose membrane lipids are largely anionic. As a consequence it can be predicted which bacterial species will be most affected by antimicrobial agents that function principally by this mechanism. This finding allows for the design of new antibiotics with selective toxicity against different groups of bacteria.     

Parallel analysis of antimicrobial activities in microbial community by SSCP based on CE

Conventional antimicrobial activity analyses such as the broth dilution method and disk diffusion test are considerably demanding processes for new antimicrobial agent discovery and sensitive diagnosis of infectious diseases. Here, we developed a new antimicrobial activity analysis system using CE-based SSCP (CE-SSCP) combined with 16S rRNA gene-specific PCR (PCR/CE-SSCP). Using this method, the population change in the microbial community in response to specific antimicrobial agents could be quantified with a high sensitivity and accuracy from a small sample amount. Using a mixture of microorganisms comprising Escherichia coli, Corynebacterium glutamicum, Acinetobacter calcoaceticus, and Staphylococcus aureus as a model system, the linear correlation between the genomic DNA concentrations and peak areas in 16S rRNA gene-specific PCR/CE-SSCP was determined; consequently, quantification of cell concentrations could be demonstrated using this method. Compared to the minimum inhibitory concentration (MIC) values from the conventional broth dilution method, this new system provided almost the same MIC values for popular antimicrobial agents such as kanamycin, spectinomycin, and streptomycin. The results demonstrated that the newly developed method can be a substitute for the conventional antimicrobial analysis method and highlighted its high potential in the areas of new antimicrobial agent discovery and clinical diagnosis.     

A cancer cell turn-on protein-CuSMn nanoparticle as the sensor of breast cancer cell and CH3O-PEG-phosphatide

A cancer activated protein-inorganic nanoparticle was used as breast cancer cell turn-on fluorescence sensor and NIR activated attenuator.     

Discovery of ferrocene-carborane derivatives as novel chemical antimicrobial agents against multidrug-resistant bacteria

Antimicrobial resistance has now become a very serious global public health problem. New drug discovery and development are urgently needed to combat the growing threat of multidrug-resistant (MDR) bacteria. The aim of this study was to explore the potential application of three ferrocene-carborane derivatives as new promising agents to confront the problem of increasing antibiotic resistance. The results of agar diffusion bioassay, minimal inhibitory concentrations (MIC) testing and time-kill assay illustrate their broad-spectrum antimicrobial activities to both American Type Culture Collection (ATCC) control strains and MDR clinical isolates. It is evident that the relevant antimicrobial properties are all in a dose-dependent manner and gradually transform into a bactericidal effect from a bacteriostatic effect with the increasing of the drug concentration. Furthermore, these ferrocene-carborane derivatives have no/little toxic effect on normal cells like HELF cells and lead to little hemolysis at their MICs. This raises the possibility to develop novel antimicrobial drugs using these new ferrocene carborane derivants.     

Lights and Shadows on the Therapeutic Use of Antimicrobial Peptides

The emergence of antimicrobial-resistant infections is still a major concern for public health worldwide. The number of pathogenic microorganisms capable of resisting common therapeutic treatments are constantly increasing, highlighting the need of innovative and more effective drugs. This phenomenon is strictly connected to the rapid metabolism of microorganisms: due to the huge number of mutations that can occur in a relatively short time, a colony can “adapt” to the pharmacological treatment with the evolution of new resistant species. However, the shortage of available antimicrobial drugs in clinical use is also caused by the high costs involved in developing and marketing new drugs without an adequate guarantee of an economic return; therefore, the pharmaceutical companies have reduced their investments in this area. The use of antimicrobial peptides (AMPs) represents a promising strategy for the design of new therapeutic agents. AMPs act as immune defense mediators of the host organism and show a poor ability to induce antimicrobial resistance, coupled with other advantages such as a broad spectrum of activity, not excessive synthetic costs and low toxicity of both the peptide itself and its own metabolites. It is also important to underline that many antimicrobial peptides, due to their inclination to attack cell membranes, have additional biological activities, such as, for example, as anti-cancer drugs. Unfortunately, they usually undergo rapid degradation by proteolytic enzymes and are characterized by poor bioavailability, preventing their extensive clinical use and landing on the pharmaceutical market. This review is focused on the strength and weak points of antimicrobial peptides as therapeutic agents. We give an overview on the AMPs already employed in clinical practice, which are examples of successful strategies aimed at overcoming the main drawbacks of peptide-based drugs. The review deepens the most promising strategies to design modified antimicrobial peptides with higher proteolytic stability with the purpose of giving a comprehensive summary of the commonly employed approaches to evaluate and optimize the peptide potentialities.     

CellFacts II – single cell analysis in real time

CellFacts II integrates electrical flow impedance and fluorescence to determine the number, size and fluorescence characteristics of individual cells in a conductive fluid. The instrument has been optimised to detect and enumerate viable and non-viable cells in fluid samples with varied particulate content, i.e. total viable counts, with discrimination of the physiological status of the individual cells. The study shows the analysis of the physiological state of individual cells in a population, effectively in real-time, enabling the rapid determination of the effect of antimicrobial agents on these cells i.e. rapid determination and optimisation of antimicrobial agents in aqueous paint systems.     

DCAP: a broad-spectrum antibiotic that targets the cytoplasmic membrane of bacteria

Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria. Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial biofilms. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents.     

Increased In Vitro Lysosomal Function in Oxidative Stress-Induced Cell Lines

Exposure of mammalian cells to oxidative stress alters lysosomal enzymes. Through cytochemical analysis of lysosomes with LysoTracker, we demonstrated that the number and fluorescent intensity of lysosome-like organelles in HeLa cells increased with exposure to hydrogen peroxide (H₂O₂), 6-hydroxydopamine (6-OHDA), and UVB irradiation. The lysosomes isolated from HeLa cells exposed to three oxidative stressors showed the enhanced antimicrobial activity against Escherichia coli. Further, when lysosomes that were isolated from HeLa cells exposed by oxidative stress were treated to normal HeLa cells, the viability of the HeLa cells was drastically reduced, suggesting increased in vitro lysosomal function (i.e., antimicrobial activity, apoptotic cell death). In addition, we also found that cathepsin B and D were implicated in increased in vitro lysosomal function when isolated from HeLa cells exposed by oxidative stress. Decrease in cathepsin B activity and increase in cathepsin D activity were observed in lysosomes isolated from HeLa cells after treatment with H₂O₂, 6-ODHA, or UVB, but cathepsin B and D were not the sole factors to induce cell death by in vitro lysosomal function. Therefore, these studies suggest a new approach to use lysosomes as antimicrobial agents and as new materials for treating cancer cell lines.     

Versatile Nanodelivery Platform to Maximize siRNA Combination Therapy

The unsatisfactory outcomes of typical multiple cytotoxic chemotherapeutic combination therapies used to treat patients have fostered a need for new unconventional combinations of therapeutic agents. Among the candidates, siRNA has been widely discussed and tested. However, the right time right place codelivery of siRNA with other types of active ingredients is challenging because of the possible differences among their physiochemical and pharmacodynamics properties. To accomplish a synergistic cytotoxic effect, a nanoassembly is thus designed to codeliver siRNA with other therapeutic agents. A siRNA, targeting prosurvival gene for the p75 neurotrophin receptor, and an organelle‐fusing peptide, targeting mitochondria, are layered onto a nanotemplate by charge–charge interaction, followed by a layer of CD44 targeting ligand. The formulated triple‐functional nanomedicine is efficiently internalized by the CD44 expressing triple‐negative breast cancer cells. The encapsulated siRNA and the pro‐apoptotic peptide are released inside cells, silencing the intended prosurvival gene, and inducing apoptosis by fusing the mitochondrial membrane, respectively. A synergistic effect is achieved by this three‐agent combination. The design of the developed multifunctional nanomedicine can be generalized to deliver other siRNA and drugs for a maximum therapeutic combination with minimal off‐targeting effects.

     

具有荧光探针的靶向肽/聚(醚-酰胺)制备及其肝癌细胞靶向性能研究

通过端氨基聚乙二醇PEG(Ⅰ)与二亚乙基三胺五乙酸二酐(DTPAA)开环合成新型端氨基聚(醚-酰胺)(PEG/DTPA)共聚物造影剂配体(Ⅱ)(Step1);Ⅱ的端氨基与偶联剂3-马来酰亚胺苯甲酸-N-琥珀酰亚胺酯(MBS)的活化端COOH反应,生成偶联剂/聚(醚-酰胺)MB/PEG/DTPA(Ⅲ′)(Step2);再通过Ⅲ′中MBS的CC双键与肝癌细胞靶向黏附肽FAM-AGKGTPSLETTPC-(SH)-COOH(FAM-13)上的巯基SH发生Michael加成反应(Step3),合成含有荧光探针FAM(5-carboxyfluorescein)的肝癌靶向肽/聚(醚-酰胺)(FAM-13/PEG/DTPA,Ⅲ).用1H-NMR和13C-NMR等方法对共聚物进行表征.Ⅲ对正常肝细胞L-02几乎观察不到荧光现象,而对肝癌细胞BEL-7404则有很强的黄绿色荧光,Ⅲ对肝癌细胞有很强的靶向性.大分子配体Ⅲ可望用于制备大分子造影剂及靶向载体负载药物.