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.     

Approaches for Mitigating Microbial Biofilm-Related Drug Resistance: A Focus on Micro- and Nanotechnologies

Biofilms play an essential role in chronic and healthcare-associated infections and are more resistant to antimicrobials compared to their planktonic counterparts due to their (1) physiological state, (2) cell density, (3) quorum sensing abilities, (4) presence of extracellular matrix, (5) upregulation of drug efflux pumps, (6) point mutation and overexpression of resistance genes, and (7) presence of persister cells. The genes involved and their implications in antimicrobial resistance are well defined for bacterial biofilms but are understudied in fungal biofilms. Potential therapeutics for biofilm mitigation that have been reported include (1) antimicrobial photodynamic therapy, (2) antimicrobial lock therapy, (3) antimicrobial peptides, (4) electrical methods, and (5) antimicrobial coatings. These approaches exhibit promising characteristics for addressing the impending crisis of antimicrobial resistance (AMR). Recently, advances in the micro- and nanotechnology field have propelled the development of novel biomaterials and approaches to combat biofilms either independently, in combination or as antimicrobial delivery systems. In this review, we will summarize the general principles of clinically important microbial biofilm formation with a focus on fungal biofilms. We will delve into the details of some novel micro- and nanotechnology approaches that have been developed to combat biofilms and the possibility of utilizing them in a clinical setting.     

Bright, “Clickable” Porphyrins for the Visualization of Oxygenation under Ambient Light

A new group of “clickable” and brightly emissive metalloporphyrins has been developed for the visualization of oxygenation under ambient light with the naked eye. These alkynyl‐terminated compounds permit the rapid and facile synthesis of oxygen‐sensing dendrimers through azide–alkyne click chemistry. With absorption maxima overlapping with the wavelengths of common commercial laser sources, they are readily applicable to biomedical imaging of tissue oxygenation. An efficient synthetic methodology, featuring the stable trimethylacetyl (pivaloyl) protecting group, is described for their preparation. A paint‐on liquid bandage containing a new, click‐synthesized porphyrin dendrimer has been used to map oxygenation across an ex vivo porcine skin burn model.     

Optimal photosensitizers for photodynamic therapy of infections should kill bacteria but spare neutrophils

Photodynamic therapy (PDT) for localized microbial infections exerts its therapeutic effect both by direct bacterial killing and also by the bactericidal effects of host neutrophils stimulated by PDT. Therefore, PDT-induced damage to neutrophils must be minimized, while direct photoinactivation of bacteria is maintained to maximize the therapeutic efficacy of antimicrobial PDT in vivo. However, there has been no study in which the cytocidal effect of PDT on neutrophils was investigated. In this study, the cytocidal effects of PDT on neutrophils were evaluated using different antimicrobial photosensitizers to find suitable candidate photosensitizers for antimicrobial PDT. PDT on murine peripheral-blood neutrophils was performed in vitro using each photosensitizer at a concentration that exerted a maximum bactericidal effect on methicillin-resistant Staphylococcus aureus, and morphological alteration and viability of neutrophils were studied. Most neutrophils were viable (>80%) after PDT using toluidine blue-O (TB) or methylene blue (MB), while neutrophils showed morphological change and their viabilities were decreased (<70%) after PDT using other photosensitizers (erythrosine B, rose bengal, crystal violet, Photofrin, new methylene blue and Laserphyrin). These results suggest that PDT using TB or MB can preserve host neutrophils while exerting a significant therapeutic effect on in vivo localized microbial infection.     

Quaternized and Thiazole‐Functionalized Free Radical‐Generating Organometallic Dendrimers as Antimicrobial Platform against Multidrug‐Resistant Microorganisms

New macromolecules such as dendrimers are increasingly needed to drive breakthroughs in diverse areas, for example, healthcare. Here, the authors report hybrid antimicrobial dendrimers synthesized by functionalizing organometallic dendrimers with quaternary ammonium groups or 2‐mercaptobenzothiazole. The functionalization tunes the glass transition temperature and antimicrobial activities of the dendrimers. Electron paramagnetic resonance spectroscopy reveals that the dendrimers form free radicals, which have significant implications for catalysis and biology. In vitro antimicrobial assays indicate that the dendrimers are potent antimicrobial agents with activity against multidrug‐resistant pathogens such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus faecium as well as other microorganisms. The functionalization increases the activity, especially in the quaternary ammonium group‐functionalized dendrimers. Importantly, the activities are selective because human epidermal keratinocytes cells and BJ fibroblast cells exposed to the dendrimers are viable after 24 h.

     

Host defense peptides and lipopeptides: modes of action and potential candidates for the treatment of bacterial and fungal infections

Endogenous peptide antibiotics (termed also host-defense or antimicrobial peptides) are known as evolutionarily old components of innate immunity. They were found initially in invertebrates, but later on also in vertebrates, including humans. This secondary, chemical immune system provides organisms with a repertoire of small peptides that act against invasion (for both offensive and defensive purposes) by occasional and obligate pathogens. Each antimicrobial peptide has a broad but not identical spectrum of antimicrobial activity, predominantly against bacteria, providing the host maximum coverage against a rather broad spectrum of microbial organisms. Many of these peptides interact with the target cell membranes and increase their permeability, which results in cell lysis. A second important family includes lipopeptides. They are produced in bacteria and fungi during cultivation on various carbon sources, and possess a strong antifungal activity. Unfortunately, native lipopeptides are non-cell selective and therefore extremely toxic to mammalian cells. Whereas extensive studies have emerged on the requirements for a peptide to be antibacterial, very little is known concerning the parameters that contribute to antifungal activity. This review summarizes recent studies aimed to understand how antimicrobial peptides and lipopeptides select their target cell. This includes a new group of lipopeptides highly potent against pathogenic fungi and yeast. They are composed of inert cationic peptides conjugated to aliphatic acids with different lengths. Deep understanding of the molecular mechanisms underlying the differential cells specificity of these families of host defense molecule is required to meet the challenges imposed by the life-threatening infections.     

New-generation biomedical materials: Peptide dendrimers and their application in biomedicine

Peptide dendrimers are attractive synthetic polymers and have been widely used as a new generation of biomaterials in recent years. Peptide dendrimers, as well as general dendrimers, may be synthesized to reach nano sizes, and display well-defined architectures, highly-branched structures, high density of functional terminal groups, and controllable molecular weights. On the other hand, peptide dendrimers have properties similar to proteins and some special characteristics, such as good biocompatibility, water solubility and resistance to proteolytic digestion. Due to these advantages, peptide dendrimers have received considerable attention in biomedicine. This review focuses on the development of peptide dendrimers with emphasis on their applications both in diagnostics and in therapy.     

α-环糊精/聚乙二醇自组装超分子纳米药物载体

研究了一种新型超分子纳米药物载体的制备方法及其药物释放性能. 将α-环糊精(α-CD)穿入肉桂酸改性的PEG分子链形成包含复合物(inclusion complex, IC), 通过超分子自组装成为纳米粒子. 将抗肿瘤药物阿霉素负载到纳米粒子中, 研究药物释放行为及其对肿瘤细胞的抑制效果. 以核磁共振(1H NMR)、X射线衍射(XRD)、紫外吸收光谱(UV)、动态光散射(DLS)、扫描电镜(SEM)、透射电镜(TEM)和原子力显微镜(AFM)表征了纳米粒子的结构和形貌, 用激光共聚焦显微镜(Confocal)研究了载药纳米粒子在细胞内的分布及其对肿瘤细胞的抑制效果. 结果显示超分子纳米粒子具有很好的生物相容性和药物缓释作用, 载药纳米粒子对肿瘤细胞具有很好的杀伤效果.     

Inverse Polyamidoamine (i-PAMAM) Dendrimer Antimicrobials

Here we redesigned the branches of polyamidoamine (PAMAM) dendrimers by moving the amide carbonyl group on the other side of the amide nitrogen atom, transforming the β-alaninyl-amidoethylamine branch, which easily undergoes retro-Michael reactions and renders PAMAMs intrinsically unstable, into a more stable glycyl-amidopropylamine branch. The resulting inverse PAMAM (i-PAMAM) dendrimers have the same carbon framework as PAMAMs and only differ by the position of the carbonyl group. In contrast to PAMAMs which are prepared in solution and are difficult to purify, we synthesize i-PAMAMs using high-temperature solid-phase peptide synthesis by iterative coupling and deprotection of the commercially available N,N-bis(N′-Fmoc-3-aminopropyl)glycine and purify them preparative reverse phase HPLC. Our i-PAMAM dendrimers show no detectable degradation over time. We demonstrate this new class of dendrimers with the synthesis of antimicrobial dendrimers with potent yet non-membrane disruptive activities against both Gram-negative and Gram-positive bacteria.     

Unraveling dendrimer translocation across cell membrane mimics

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.     

Peptide and glycopeptide dendrimer apple trees as enzyme models and for biomedical applications

Solid phase peptide synthesis (SPPS) provides peptides with a dendritic topology when diamino acids are introduced in the sequences. Peptide dendrimers with one to three amino acids between branches can be prepared with up to 38 amino acids (MW ~ 5,000 Da). Larger peptide dendrimers (MW ~ 30,000) were obtained by a multivalent chloroacetyl cysteine (ClAc) ligation. Structural studies of peptide dendrimers by CD, FT-IR, NMR and molecular dynamics reveal molten globule states containing up to 50% of α-helix. Esterase and aldolase peptide dendrimers displaying dendritic effects and enzyme kinetics (k(cat)/k(uncat) ~ 10(5)) were designed or discovered by screening large combinatorial libraries. Strong ligands for Pseudomonas aeruginosa lectins LecA and LecB able to inhibit biofilm formation were obtained with glycopeptide dendrimers. Efficient ligands for cobalamin, cytotoxic colchicine conjugates and antimicrobial peptide dendrimers were also developed showing the versatility of dendritic peptides. Complementing the multivalency, the amino acid composition of the dendrimers strongly influenced the catalytic or biological activity obtained demonstrating the importance of the "apple tree" configuration for protein-like function in peptide dendrimers.     

Inhibition of Quorum Sensing,Motility and Biofilm Formation of Pseudomonas aeruginosa by Copper Oxide Nanostructures

Quorum sensing (QS) is the communication between bacterial cells governed by their population density and regulated by the genes controlling virulence factors and biofilm formation. Multiple mechanisms of biofilms are resistive to antimicrobial chemotherapy; therefore novel strategies are required to overcome its limitations. Here, we report the effect of various copper oxide nanostructures (CuO-NSs) on quorum sensing inhibition. The two-dimensional CuO-NSs such as interlaced nanodiscs, nanodiscs and leaf-shaped nanosheets are prepared via a simple chemical method. The Quorum sensing inhibition (QSI) activity of all the CuO-NS are examined using reporter strain Chromobacterium violaceum CV026 and Escherichia coli pSB1142. We found that the CuO-interlaced nanodisc structures exhibit better QSI activity than nanodiscs and leaf-shaped sheets. The interlaced nanodisc structures are inhibited various long-chain N-acyl homoserine lactones (AHLs) mediated QS individually and confirmed by other QS-associated phenomena for Pseudomonas aeruginosa, including biofilm inhibition, inhibition of virulence factors such as pyocyanin, protease production and swarming motility. Thus QSI activity of CuO-NSs is solely dependent on specific shape offering large surface area and more active sites. The CuO-NS is effective quorum sensing inhibitors, which has potential clinical applications in the management of P. aeruginosa associated infections.

     

Supramolecular fluorophores for biological studies: phenylene vinylene-amino acid amphiphiles

We report here on a family of self-assembling fluorescent organic amphiphiles with a biomolecular L-lysine hydrophile and a photonically active phenylene vinylene hydrophobe. Unlike conventional amphiphiles, these segmented dendrimers feature a rigid, branched hydrophobe, and have packing characteristics controlled by the ratio of cross-sectional areas of the hydrophobe and hydrophile. In dilute solution, the amphiphiles form supramolecular aggregates, which are easily taken in by cells through an endocytic pathway, and have no discernible effect on cell proliferation or morphology. An analogous pyrene-based amphiphile was cytotoxic, suggesting that cell survival may be linked either to the self-assembling nature of the amphiphiles, or to the specific properties of the phenylene vinylene segment. The combination of photonic and biological components in these amphiphiles provides great potential for applications in sensing or delivery of molecules to intracellular targets.     

Adhesive peptides conjugated PAMAM dendrimer as a coating polymeric material enhancing cell responses

This present work aims to functionalize poly(amidoamine) (PAMAM) dendrimers with various reported adhesive peptides, including Arg-Gly-Asp (RGD), Tyr-Ile-Gly-Ser-Arg (YIGSR), and Ile-Lys-Val-Ala-Val (IKVAV) for enhancing cell responses. The RGD, YIGSR, or IKVAV functionalized PAMAM coated substrate could promote cell adhesion of bone marrow mesenchymal stem cells (BMSCs) within 1 h after incubation. The neurite differentiation and proliferation of pheochromocytoma (PC12) cells were also significantly enhanced after culturing on the peptide functionalized PAMAM dendrimers for two and four days. This peptide functionalized PAMAM dendrimers are considered as the potential candidates for various tissue engineering applications.     

Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are used by over 70 Gram-negative bacterial species as autoinducers. Inhibition of QS signaling might represent a new target for antimicrobial therapy. Here we report the hapten design, synthesis, generation of monoclonal antibodies (mAbs) against AHLs, and the evaluation of these mAbs for their ability to blunt QS signaling and inhibit virulence factor expression in P. aeruginosa. The mAbs can be envisioned as a tool for future investigations into AHL-based QS, which may aid in gaining new insights into the pathogenesis of P. aeruginosa and may ultimately lead to the development of new strategies to combat bacterial diseases.     

Microbial interactions in <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> biofilms

Medical device-associated infections, most frequently caused by coagulase-negative staphylococci, especially Staphylococcus epidermidis, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is the most important factor in the pathogenesis of these infections, which regularly fail to respond to appropriate antimicrobial therapy. Progress in elucidating the factors functional in elaboration of S. epidermidis biofilms and the regulation of their expression with a special emphasis on the role of quorum sensing are reviewed. Significant progress has been made in recent years, which provides the rationale for developing better preventive, therapeutic and diagnostic measures.     

Evaluation of bactericidal and fungicidal activity of ferrocenyl or phenyl derivatives in the diphenyl butene series

Some ferrocene compounds, such as tamoxifen derivatives hydroxyferrocifen 1 and ferrociphenol 2, show strong antiproliferative activity on hormone-dependent and hormone-independent breast cancer cells. In order to evaluate their antimicrobial activity, they were tested, together with their purely organic analogs, on the bacteria Pseudomonas aeruginosa and Staphylococcus aureus and the fungus Candida albicans. It has been found that the compounds bearing alkylamino chains are active, and in these cases the antimicrobial activity increases for compounds bearing two amino chains. These dialkyamino compounds are equally as active as doxycycline on P. aeruginosa and S. aureus but superior to it on C. albicans. The results show that there are no general correlation between the antitumoral activity and the bactericidal and fungicidal activities of these compounds. The ferrocene derivatives and their organic analogs have similar activity on bacteria and fungus. This bactericidal and fungicidal behaviour is a novel area of activity for these entities.     

Peptide-functionalized polyphenylene dendrimers

This contribution describes the synthesis of polyphenylene dendrimers that are functionalized with up to 16 lysine residues or substituted with short peptide sequences composed of 5 lysine or glutamic acid repeats and a C- or N-terminal cysteine residue. Polyphenylene dendrimers were prepared via a sequence of Diels-Alder cycloaddition and deprotection reactions from cyclopentadienone building blocks. Single amino acids could be introduced on the periphery of the dendrimers by using amino acid substituted cyclopentadienones in the last Diels-Alder addition reaction. Alternatively, peptide sequences were attached via a chemoselective reaction, which involved the addition of the sulfhydryl group of a cysteine residue of an oligopeptide to a maleimide moiety present on the surface of the dendrimer. These amino acid and peptide functionalized dendrimers may be of interest as model compounds to study DNA complexation and condensation or as building blocks for the preparation of novel supramolecular architectures via layer-by-layer self-assembly.     

Intracellular antimicrobial photodynamic therapy: a novel technique for efficient eradication of pathogenic bacteria

The increasing resistance of bacteria to antibiotics is a serious problem, caused in part by excessive and improper use of these drugs. One alternative to traditional antibiotic therapy is photodynamic antimicrobial chemotherapy (PACT) which is based on the use of a photosensitizer (PS), activated by illumination with visible light. The poor penetration of visible light through the skin limits the application of PACT to the treatment of skin infections or the use of invasive procedures. To overcome this problem we report the exploitation of light emitted as a result of the chemiluminescent reaction of luminol to excite the PS and we call this process chemiluminescent photodynamic antimicrobial therapy (CPAT). We studied the effect of free and liposome-encapsulated PS (methylene blue or toluidine blue) on bacteria under excitation by either white external light or chemiluminescence emitted by free or liposome-enclosed luminol. PACT showed slightly better performance that CPAT for free and encapsulated PS for both types of bacteria. CPAT resulted in a three log suppression of Staphylococcus aureus and two log suppression of Escherichia coli growth. The use of CPAT may prove to be a novel and more effective form of antimicrobial therapy, particularly for internal infections not easily accessible to traditional PACT.     

Recent advances in antimicrobial surfaces via tunable molecular interactions: Nanoarchitectonics and bioengineering applications

Infections can lead to severe health issues, even death. Surfaces, such as those of biomedical devices, implants, textiles, tables and doorknobs, play a crucial role as carriers for pathogens to migrate, attach and proliferate. Implementing surfaces with antimicrobial properties offers a reliable and long-lasting approach to combat surface transmission of germs, minimize microbial colonization, and reduce infections. In this review, we present recent advancements in antimicrobial surfaces, categorized into four groups based on their action mechanisms: antifouling, bactericidal, antifouling and bactericidal, and dynamic or stimuli-responsive surfaces. The work highlights the fabrication processes and properties of each category, along with discussing their structure-performance relationships. Special attention is given to various anchoring strategies involving tunable molecular interactions. The review also introduces relevant biomedical applications.