Zinc(II) Iminopyridine Complexes as Antibacterial Agents: A Structure-to-Activity Study.

Antibiotic resistance is currently a global health emergency. Metallodrugs, especially metal coordination complexes, comprise a broad variety of candidates to combat antibacterial infections. In this work, we designed a new family of Schiff base zinc(II) complexes with iminopyridine as an organic ligand and different inorganic ligands: chloride, nitrate, and acetate. The antibacterial effect of the Zn(II) complexes was studied against planktonic bacterial cells of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) strains. The results showed a moderate biocide activity in both types of planktonic bacteria, which arises from the metal complexation to the Schiff base ligand. Importantly, we confirmed the crucial effect of the metal, with Zn(II) improving the activity of Cu(II) counterparts previously reported. On the other hand, the impact of the inorganic ligands was not significant for the antibacterial effect but was relevant for the complex solubility. Finally, as proof of concept of topical antibacterial formulation, we formulated an emulsion containing the most lipophilic Zn(II) complex and confirmed a sustained release for 24 h in a vertical cell diffusion assay. The promising activity of iminopyridine Zn(II) complexes is potentially worth exploring in more detailed studies.

Bifunctional Carbosilane Dendrimers for the Design of Multipurpose Hydrogels with Antibacterial Action.

The emergence of antibiotic resistance is a serious global health problem. There is an incessant demand for new antimicrobial drugs and materials that can address this global issue from different angles. Dendritic hydrogels have appeared as a promising strategy. A family of bifunctional amphiphilic carbosilane dendrimers was designed and employed as nanosized cross-linking points for the synthesis of high-swelling hydrogels using the highly efficient Thiol-Ene click reaction for their preparation. Both stoichiometric and off-stoichiometric conditions were studied, generating hydrogels with pendant hydroxyl or alkene moieties. These hydrogels were found to be tunable antibacterial materials. They can easily be postmodified with relevant antibiotic moieties through covalent attachment on the hydroxyl or alkene pendant groups, generating ammonium-decorated networks with temperature and pH-responsive properties. Additionally, they can efficiently encapsulate drugs with poor solubility in water, like ciprofloxacin, and perform a sustained release over time, as demonstrated in preliminary assays against Staphylococcus aureus.

Potential anti-adhesion activity of novel carbosilane zwitterionic dendrimers against eukaryotic and prokaryotic pathogenic microorganisms.

The development of biofilms on different surfaces continues to be a major public health problem. The antimicrobial resistance and the difficulty of finding drugs capable of combating these established biofilms generates the urgent need to find compounds that prevent cells from settling and establishing of these complex communities of microorganisms. Zwitterionic modification of nanomaterials allows the formation of a hydration layer, and this highly hydrophilic surface provides antifouling properties as well as a good biocompatibility by preventing non-specific interactions. Thus, they are appropriate candidates to prevent microbial adhesion to different surfaces and, in consequence, avoid biofilm formation. For this reason, we have incorporated zwitterionic moieties in multivalent systems, as are carbosilane dendrimers. Characterization of these systems was performed using nuclear magnetic resonance and mass spectrometry. It has been analysed if the new molecules have capacity to inhibit the biofilm formation in Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that they were more effective against S. aureus, observing a biofilm reduction of 81.5% treating with 32 mg/L of G2SiZWsf dendrimer and by 72.5% using 32 mg/L of the G3SiZWsf dendrimer. Finally, the absence of cytotoxicity was verified by haemolysis and cytotoxicity studies in human cells lines.

Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays.

Background: In vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity. Methods: The viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy. Results: It was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity. Discussion: Our findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.

Inhibition of Candida glabrata Biofilm by Combined Effect of Dendritic Compounds and Amphotericin.

In the last decade, Candida glabrata has become an important emerging opportunistic pathogen not only because of the increase in nosocomial infections frequency but also because of its ability to form biofilms and its innate resistance to commercial antifungals. These characteristics make this pathogen a major problem in hospital settings, including problems regarding equipment, and in immunosuppressed patients, who are at high risk for candidemia. Therefore, there is an urgent need for the development of and search for new antifungal drugs. In this study, the efficacy of two dendritic wedges with 4-phenyl butyric acid (PBA) at the focal point and cationic charges on the surface ArCO2G2(SNMe3I)4 (1) and ArCO2G3(SNMe3I)8 (2) was studied against C. glabrata strain to inhibit the formation of biofilms and eliminate established biofilm. For this, MBIC (minimum biofilm inhibitory concentration), MBDC (minimum biofilm damaging concentrations), as well as MFCB (minimum fungicidal concentration in biofilm) and MBEC (minimum biofilm eradicating concentration) were determined. In addition, different combinations of dendrons and amphotericin B were tested to study possible synergistic effects. On the other hand, cytotoxicity studies were performed. C. glabrata cells and biofilm structure were visualized by confocal microscopy. ArCO2G2(SNMe3I)4 (1) and ArCO2G3(SNMe3I)8 (2) dendrons showed both an MBIC of 8 mg/L and a MBDC of 32 mg/L and 64 mg/L, respectively. These dendrons managed to eradicate the entirety of an established biofilm. In combination with the antifungal amphotericin, it was possible to prevent the generation of biofilms and eradicate established biofilms at lower concentrations than those required individually for each compound at these conditions.

Eradication of Candida albicans Biofilm Viability: In Vitro Combination Therapy of Cationic Carbosilane Dendrons Derived from 4-Phenylbutyric Acid with AgNO3 and EDTA.

Candida albicans is a human pathogen of significant clinical relevance. This pathogen is resistant to different drugs, and most clinical antifungals are not effective against the prevention and treatment of C. albicans infections. As with other microorganisms, it can produce biofilms that serve as a barrier against antifungal agents and other substances, contributing to infection in humans and environmental tolerance of this microorganism. Thus, resistances and biofilm formation make treatment difficult. In addition, the complete eradication of biofilms in implants, catheters and other medical devices, is challenging and necessary to prevent relapses of candidemia. Therefore, it is a priority to find new molecules or combinations of compounds with anti-Candida biofilm activity. Due to the difficulty of treating and removing biofilms, the aim of this study was to evaluate the in vitro ability of different generation of cationic carbosilane dendrons derived from 4-phenylbutyric acid, ArCO2Gn(SNMe3I)m, to eradicate C. albicans biofilms. Here, we assessed the antifungal activity of the second generation dendron ArCO2G2(SNMe3I)4 against C. albicans cells and established biofilms since it managed to seriously damage the membrane. In addition, the combinations of the second generation dendron with AgNO3 or EDTA eradicated the viability of biofilm cells. Alterations were observed by scanning electron microscopy and cytotoxicity was assessed on HeLa cells. Our data suggest that the dendritic compound ArCO2G2(SNMe3I)4 could represent an alternative to control the infections caused by this pathogen.

Effect of the Combination of Levofloxacin with Cationic Carbosilane Dendron and Peptide in the Prevention and Treatment of Staphylococcus aureus Biofilms.

Antibiotic resistance and biofilm-related infections, persistent in conventional antimicrobial treatment, are continuously increasing and represent a major health problem worldwide. Therefore, the development of new effective treatments to prevent and treat biofilm-related infections represents a crucial challenge. Unfortunately, the extensive use of antibiotics has led to an increase of resistant bacteria with the subsequent loss of effectivity of commercial antibiotics, mainly due to antibiotic resistance and the ability of some bacteria to form microbial communities in biotic or abiotic surfaces (biofilms). In some cases, these biofilms are resistant to high concentrations of antibiotics that lead to treatment failure and recurrence of the associated infections. In the fight against microbial resistance, the combination of traditional antibiotics with new compounds (combination therapy) is an alternative that is becoming more extensive in the medical field. In this work, we studied the cooperative effects between levofloxacin, an approved antibiotic, and peptides or cationic dendritic molecules, compounds that are emerging as a feasible solution to overcome the problem of microbial resistance caused by pathogenic biofilms. We studied a new therapeutic approach that involves the use of levofloxacin in combination with a cationic carbosilane dendron, called MalG2(SNHMe2Cl)4, or a synthetic cell-penetrating peptide, called gH625, conjugated to the aforementioned dendron. To carry out the study, we used two combinations (1) levofloxacin/dendron and (2) levofloxacin/dendron-peptide nanoconjugate. The results showed the synergistic effect of the combination therapy to treat Staphylococcus aureus biofilms. In addition, we generated a fluorescein labeled peptide that allowed us to observe the conjugate (dendron-peptide) localization throughout the bacterial biofilm by confocal laser scanning microscopy.

In Vitro Evaluation of the Combination of Melaleuca alternifolia (Tea Tree) Oil and Dimethyl Sulfoxide (DMSO) against Trophozoites and Cysts of Acanthamoeba Strains. Oxygen Consumption Rate (OCR) Assay as a Method for Drug Screening.

Ameobae belonging to the genus Acanthamoeba are responsible for the human diseases Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage (cysts). In an attempt to add new agents that are effective against trophozoites and cysts, tea tree oil (TTO) and dimethyl sulfoxide (DMSO), separately and in combination, were tested In Vitro against two Acanthamoeba isolates, T3 and T4 genotypes. The oxygen consumption rate (OCR) assay was used as a drug screening method, which is to some extent useful in amoebicide drug screening; however, evaluation of lethal effects may be misleading when testing products that promote encystment. Trophozoite viability analysis showed that the effectiveness of the combination of both compounds is higher than when either compound is used alone. Therefore, the TTO alone or TTO + DMSO in combination were an amoebicide, but most of the amoebicidal activity in the combination's treatments seemed to be caused mainly by the TTO effect. In fact, DMSO alone seems to be a non-amoebicide, triggering encystment. Regarding cytotoxicity, these compounds showed toxicity in human corneal epithelial cells (HCEpiC), even at low concentrations when tested in combination. In conclusion, the use of TTO and DMSO, in combination or alone, cannot be recommended as an alternative for AK treatment until more cytotoxicity and cyst adhesion tests are performed.

In Vitro Activity of Carbosilane Cationic Dendritic Molecules on Prevention and Treatment of Candida Albicans Biofilms.

Candida spp. are one of the most common fungal pathogens. Biofilms formed by Candidaalbicans offer resistance mechanisms against most antifungal agents. Therefore, development of new molecules effective against these microorganisms, alone or in combination with antifungal drugs, is extremely necessary. In the present work, we carried out a screening process of different cationic carbosilane dendritic molecules against C. albicans. In vitro activity against biofilm formation and biofilms was tested in both Colección Española de Cultivos Tipo (CECT) 1002 and clinical C. albicans strains. Cytotoxicity was studied in human cell lines, and biofilm alterations were observed by scanning electron microscopy (SEM). Antifungal activity of the carbosilane dendritic molecules was assessed by monitoring cell viability using both established and novel cell viability assays. One out of 14 dendritic molecules tested, named BDSQ024, showed the highest activity with a minimum biofilm inhibitory concentration (MBIC) for biofilm formation and a minimum biofilm damaging concentration (MBDC) for existing biofilm of 16-32 and 16 mg/L, respectively. Synergy with amphotericin (AmB) and caspofungin (CSF) at non-cytotoxic concentrations was found. Therefore, dendritic compounds are exciting new antifungals effective at preventing Candida biofilm formation and represent a potential novel therapeutic agent for treatment of C. albicans infection in combination with existing clinical antifungals.

Ultrastructural Study of Acanthamoeba polyphaga Trophozoites and Cysts Treated In Vitro with Cationic Carbosilane Dendrimers.

Cationic carbosilane dendrimers are branched molecules with antimicrobial properties. Their activity has been tested against Acanthamoeba polyphaga, a causative agent of Acanthamoeba keratitis, a severe ocular disease in humans. A. polyphaga trophozoites and cysts were exposed to different noncytotoxic cationic carbosilane dendrimers with proven antiamoebic activity. The effects of treatment on cell surface and cell ultrastructure were examined by scanning and transmission electron microscopy, respectively. Two of the dendrimers tested induced dramatic alterations of cellular ultrastructure in both trophozoites and cysts, including vacuolization, depletion of cytoplasmic contents, and reduced cell size. Additionally, we observed severe alterations of the plasma membrane with membrane blebbing in trophozoites and disruption in cysts. These alterations were also observed with chlorhexidine, a drug used for treatment of Acanthamoeba keratitis. Our results support that these compounds may target membranes, and their action is critical for parasite integrity.

TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division.

Dynamin-related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP-2) complex subunit alpha-1 and a homolog of the ezrin-radixin-moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re-distribution of TgDrpC to the IMC during division is dependent on post-Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.

Evaluation of the activity of new cationic carbosilane dendrimers on trophozoites and cysts of Acanthamoeba polyphaga.

Dendrimers are repetitively branched molecules with a broad spectrum of applications, mainly for their antimicrobial properties and as nanocarriers for other molecules. Recently, our research group have synthesized and studied their activity against Acanthamoeba sp., causative agent of a severe ocular disease in humans: Acanthamoeba keratitis. New cationic carbosilane dendrimers were tested against the protozoa forms at different concentrations and for different incubation times. Trophozoite viability was determined by manual counting and cyst viability by observing excystment in microplates with fresh culture medium. Cytotoxicity was checked on HeLa cells using the microculture tetrazolium assay. Alterations were observed by optical microscopy and by flow cytometry staining with propidium iodide. Six out of the 18 dendrimers tested were non-cytotoxic and effective against the trophozoite form, having one of them (dendrimer 14 with an IC50 of 2.4 + 0.1 mg/L) a similar activity to chlorhexidine digluconate (IC50 1.7 + 0.1 mg/L). This dendrimer has a polyphenoxo core and a sulphur atom close to the six -NH3+ terminal groups. On the other hand, only two dendrimers showed some effect against cysts (dendrimers 14 and 17). However, their minimum cysticidal concentrations were cytotoxic and less effective than the control drug. The alterations on the amoeba morphology produced by the treatment with dendrimers were size reduction, increased complexity, loss of acanthopodia and cell membrane disruption. In conclusion, these results suggest that some dendrimers may be studied in animal models to test their effect and that new dendrimers with similar features should be synthesized.