Multimodal evaluation of drug antibacterial activity reveals cinnamaldehyde analog anti-biofilm effects against Haemophilus influenzae.

Biofilm formation by the pathobiont Haemophilus influenzae is associated with human nasopharynx colonization, otitis media in children, and chronic respiratory infections in adults suffering from chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). ß-lactam and quinolone antibiotics are commonly used to treat these infections. However, considering the resistance of biofilm-resident bacteria to antibiotic-mediated killing, the use of antibiotics may be insufficient and require being replaced or complemented with novel strategies. Moreover, unlike the standard minimal inhibitory concentration assay used to assess antibacterial activity against planktonic cells, standardization of methods to evaluate anti-biofilm drug activity is limited. In this work, we detail a panel of protocols for systematic analysis of drug antimicrobial effect on bacterial biofilms, customized to evaluate drug effects against H. influenzae biofilms. Testing of two cinnamaldehyde analogs, (E)-trans-2-nonenal and (E)-3-decen-2-one, demonstrated their effectiveness in both H. influenzae inhibition of biofilm formation and eradication or preformed biofilms. Assay complementarity allowed quantifying the dynamics and extent of the inhibitory effects, also observed for ampicillin resistant clinical strains forming biofilms refractory to this antibiotic. Moreover, cinnamaldehyde analog encapsulation into poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles allowed drug vehiculization while maintaining efficacy. Overall, we demonstrate the usefulness of cinnamaldehyde analogs against H. influenzae biofilms, present a test panel that can be easily adapted to a wide range of pathogens and drugs, and highlight the benefits of drug nanoencapsulation towards safe controlled release.

Lipid-based nanomedicines for the treatment of bacterial respiratory infections: current state and new perspectives.

The global threat posed by antimicrobial resistance demands urgent action and the development of effective drugs. Lower respiratory tract infections remain the deadliest communicable disease worldwide, often challenging to treat due to the presence of bacteria that form recalcitrant biofilms. There is consensus that novel anti-infectives with reduced resistance compared with conventional antibiotics are needed, leading to extensive research on innovative antibacterial agents. This review explores the recent progress in lipid-based nanomedicines developed to counteract bacterial respiratory infections, especially those involving biofilm growth; focuses on improved drug bioavailability and targeting and highlights novel strategies to enhance treatment efficacy while emphasizing the importance of continued research in this dynamic field.

A quality by design approach for the synthesis of palmitoyl-L-carnitine-loaded nanoemulsions as drug delivery systems.

Nanoemulsions (NE) are lipid nanocarriers that can efficiently load hydrophobic active compounds, like palmitoyl-L-carnitine (pC), used here as model molecule. The use of design of experiments (DoE) approach is a useful tool to develop NEs with optimized properties, requiring less experiments compared to trial-and-error approach. In this work, NE were prepared by the solvent injection technique and DoE using a two-level fractional factorial design (FFD) as model was implemented for designing pC-loaded NE. NEs were fully characterized by a combination of techniques, studying its stability, scalability, pC entrapment and loading capacity and biodistribution, which was studied ex-vivo after injection of fluorescent NEs in mice. We selected the optimal composition for NE, named pC-NEU, after analysis of four variables using DoE. pC-NEU incorporated pC in a very efficient manner, with high entrapment efficiency (EE) and loading capacity. pC-NEU did not change its initial colloidal properties stored at 4 °C in water during 120 days, nor in buffers with different pH values (5.3 and 7.4) during 30 days. Moreover, the scalability process did not affect NE properties and stability profile. Finally, biodistribution study showed that pC-NEU formulation was predominantly concentrated in the liver, with minimal accumulation in spleen, stomach, and kidneys.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

Polymethine dyes-loaded solid lipid nanoparticles (SLN) as promising photosensitizers for biomedical applications.

Polymethine dyes (PMD) have proved to be excellent candidates in the biomedical field for potential applications in both diagnostic and therapeutic. However, PMD application in biomedicine is hindered by their poor solubility and stability in physiological conditions. Therefore, the incorporation of these dyes in nanosystems could be important to prevent the formation of dye aggregates in aqueous environment and to protect their photophysical characteristics. In the present work, two PMD based on the benzoindolenine ring (bromine benzo-cyanine-C4 and bromine benzo-squaraine-C4) were incorporated into Solid Lipid Nanoparticles (SLN) to solubilize and stabilize them in aqueous solutions. Obtained SLN showed a high incorporation efficiency for both PMD (≈90%) and not only preserved their spectroscopic properties in the NIR region even under physiological conditions but also improved them. Viability assays showed good biocompatibility of both empty and loaded nanocarriers while the cellular uptake and intracellular localization showed the effective internalization in MCF-7 cells, with a partial mitochondrial localization for CY-SLN. Moreover, in vitro phototoxicity assay showed that cyanine loaded-SLN (CY-SLN) is more photoactive than the free dye.

Culturable halophilic bacteria inhabiting Algerian saline ecosystems: A source of promising features and potentialities.

This paper aims to characterize halophilic bacteria inhabiting Algerian Saline Ecosystems (Sebkha and Chott) located in arid and semi-arid ecoclimate zones (Northeastern Algeria). In addition, screening of enzymatic activities, heavy metal tolerance and antagonistic potential against phytopathogenic fungi were tested. A total of 74 bacterial isolates were screened and phylogenetically characterized using 16S rRNA gene sequencing. The results showed a heterogeneous group of microorganisms falling within two major phyla, 52 strains belonging to Firmicutes (70.2%) and 22 strains (30.8%) of γ-Proteobacteria. In terms of main genera present, the isolates were belonging to Bacillus, Halobacillus, Lentibacillus, Oceanobacillus, Paraliobacillus, Planomicrobium, Salicola, Terribacillus, Thalassobacillus, Salibacterium, Salinicoccus, Virgibacillus, Halomonas, Halovibrio, and Idiomarina. Most of the enzymes producers were related to Bacillus, Halobacillus, and Virgibacillus genera and mainly active at 10% of growing salt concentrations. Furthermore, amylase, esterase, gelatinase, and nuclease activities ranked in the first place within the common hydrolytic enzymes. Overall, the isolates showed high minimal inhibitory concentration values (MIC) for Ni2+ and Cu2+ (0.625 to 5 mM) compared to Cd2+ (0.1 to 2 mM) and Zn2+ (0.156 to 2 mM). Moreover, ten isolated strains belonging to Bacillus, Virgibacillus and Halomonas genera, displayed high activity against the pathogenic fungi (Botrytis cinerea, Fusarium oxyporum, F. verticillioides and Phytophthora capsici). This study on halophilic bacteria of unexplored saline niches provides potential sources of biocatalysts and novel bioactive metabolites as well as promising candidates of biocontrol agents and eco-friendly tools for heavy metal bioremediation.

Solid lipid nanoparticles for the delivery of anti-microbial oligonucleotides.

Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobial resistant (AMR) infections. Experimental antibacterial oligonucleotide therapeutics, such as transcription factor decoys (TFD), are a promising approach to circumvent AMR. However, the therapeutic potential of TFD is contingent upon the development of carriers that afford efficient DNA protection against nucleases and delivery of DNA to the target infection site. As a carrier for TFD, here we present three prototypes of anionic solid lipid nanoparticles that were coated with either the cationic bolaamphiphile 12-bis-tetrahydroacridinium or with protamine. Both compounds switched particles zeta potential to positive values, showing efficient complexation with TFD and demonstrable protection from deoxyribonuclease. The effective delivery of TFD into bacteria was confirmed by confocal microscopy while SLN-bacteria interactions were studied by flow cytometry. Antibacterial efficacy was confirmed using a model TFD targeting the Fur iron uptake pathway in E. coli under microaerobic conditions. Biocompatibility of TFD-SLN was assessed using in vitro epithelial cell and in vivo Xenopus laevis embryo models. Taken together these results indicate that TFD-SLN complex can offer preferential accumulation of TFD in bacteria and represent a promising class of carriers for this experimental approach to tackling the worldwide AMR crisis.

Inclusion of oligonucleotide antimicrobials in biocompatible cationic liposomes: A structural study.

HYPOTHESIS: Transcription factor decoys (TFD) are short oligonucleotides designed to block essential genetic pathways in bacteria and defeat resistant infections. TFD protection in biological fluids and their delivery to the site of infection require formulation in appropriate delivery systems. In this work, we build on a classical phosphatidylcholine/phosphatidylethanolamine (POPC/DOPE) scaffold to design TFD-loaded cationic liposomes by combining the DNA-complexing abilities of a bolaamphiphile, (1,1'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride (12-bis-THA), with the biocompatible cationic lipid ethyl-phosphatidylcholine (DPePC). The goal is to perform a structural study to determine the impact of the bolaamphiphile and TFD incorporation on the liposome structure, the capacity for TFD encapsulation, and the colloidal stability in saline media and cell culture environments. EXPERIMENTS: The systems are characterized by means of dynamic light scattering, small-angle X-ray scattering, and ζ-potential measurements, to provide a clear picture of the liposome structure. Circular dichroism (CD) spectroscopy is used to assess the compaction of the oligonucleotide in a psi form, while steady-state fluorescence and fluorescence correlation spectroscopies give insight into the entrapment rate and distribution of the TFD in the liposomes. FINDINGS: We found that the combination of the two cationic species, 12-bis-THA and DPePC, allows encapsulation of 90% of the TFD. Results of CD experiments revealed that the TFD is condensed, therefore likely protected from the lytic action of serum nucleases. Finally, the systems showed colloidal stability in aqueous dispersion with ionic strength comparable to biologically relevant media.

Polyarginine nanocapsules: A versatile nanocarrier with potential in transmucosal drug delivery.

The objective of this work was to investigate the potential utility of nanocapsules composed of an oily core decorated with a single polyarginine (PARG), or double PARG/polyacrylic acid (PAA) layer as oral peptide delivery carrier. A step-by-step formulation optimization process was designed, which involved the study of the influence of the surfactants, oils and polymer shells (PARG of different molecular weight and PAA) on the nanocapsules physicochemical properties, peptide loading efficiency, stability in simulated intestinal fluids (SIF) and capacity to enhance the permeability of the intestinal epithelium. Despite the lipophilic nature of the nanocapsules, it was possible to achieve a moderate loading of the hydrophilic model peptide salmon calcitonin and control its release in SIF, by adjusting the formulation conditions. Finally, studies in the Caco-2 epithelial cell line showed the capacity of the nanocapsules to reduce the transepithelial electric resistance of the monolayer, without compromising their viability. Overall, these properties suggest the capacity of polyarginine nanocapsules for enhancing the transport of peptides across epithelia.

Cationic liposomal vectors incorporating a bolaamphiphile for oligonucleotide antimicrobials.

Antibacterial resistance has become a serious crisis for world health over the last few decades, so that new therapeutic approaches are strongly needed to face the threat of resistant infections. Transcription factor decoys (TFD) are a promising new class of antimicrobial oligonucleotides with proven in vivo activity when combined with a bolaamphiphilic cationic molecule, 12-bis-THA. These two molecular species form stable nanoplexes which, however, present very scarce colloidal stability in physiological media, which poses the challenge of drug formulation and delivery. In this work, we reformulated the 12-bis-THA/TFD nanoplexes in a liposomal carrier, which retains the ability to protect the oligonucleotide therapeutic from degradation and deliver it across the bacterial cell wall. We performed a physical-chemical study to investigate how the incorporation of 12-bis-THA and TFD affects the structure of POPC- and POPC/DOPE liposomes. Analysis was performed using dynamic light scattering (DLS), ζ-potential measurements, small-angle x-ray scattering (SAXS), and steady-state fluorescence spectroscopy to better understand the structure of the liposomal formulations containing the 12-bis-THA/TFD complexes. Oligonucleotide delivery to model Escherichia coli bacteria was assessed by means of confocal scanning laser microscopy (CLSM), evidencing the requirement of a fusogenic helper lipid for transfection. Preliminary biological assessments suggested the necessity of further development by modulation of 12-bis-THA concentration in order to optimize its therapeutic index, i.e. the ratio of antibacterial activity to the observed cytotoxicity. In summary, POPC/DOPE/12-bis-THA liposomes appear as promising formulations for TFD delivery.

Polyarginine Nanocapsules as a Potential Oral Peptide Delivery Carrier.

We have previously reported the development of novel nanocapsules made of polyarginine (PArg) specifically designed for the delivery of small anticancer drugs into cells. Our goal, in this work, has been to investigate the potential of these nanocarriers for oral delivery of peptide anticancer drugs. To reach this objective, we chose the antitumoral peptide, elisidepsin, and evaluated the characteristics of the PArg nanocapsules in terms of drug loading capacity, stability in simulated intestinal fluids, and ability to interact with the intestinal epithelium both in vitro (Caco-2 model cell line) and in vivo. Our results suggest that elisidepsin can be effectively loaded into the nanocapsules by adjusting the formulation parameters, using a solvent displacement technique. The resulting nanocapsules were stable upon incubation in simulated intestinal fluids and had the ability to reduce, in a transient manner, the transepithelial electrical resistance of the Caco-2 cell monolayer. Confocal images also revealed that PArg nanocapsules were internalized by the monolayer without evident signs of cytotoxicity. Finally, the in vivo fluorescent imaging study illustrates the retention of the nanocapsules in the gastrointestinal tract upon oral administration. Overall, the results underline the potential interest of PArg nanocapsules as carriers for the oral administration of peptide drugs.

Halobellus ramosii sp. nov., an extremely halophilic archaeon isolated from a saline-wetland wildfowl reserve.

An extremely halophilic archaeon, strain S2FP14T, was isolated from a brine sample from the inland hypersaline lake Fuente de Piedra, a saline-wetland wildfowl reserve located in the province of Málaga in southern Spain. Colonies were red-pigmented and the cells were Gram-staining-negative, motile and pleomorphic. S2FP14T was able to grow in media containing 12.5-30 % (w/v) total salts (optimum 20 %) at pH 7-8.5 (optimum 7.5) and at 25-50 °C (optimum 37 °C). The 16S rRNA gene sequence analysis indicated that this strain represented a member of the genus Halobellus. S2FP14T showed a similarity of 99.5 % to Halobellus inordinatus YC20T, 96.1 % to Halobellus litoreus GX31T, 95.9 % to Halobellus limi TBN53T, 95.5 % to Halobellus rarus YC21T, 95.2 % to Halobellus rufus CBA1103T, 94.6 % to Halobellus salinus CSW2.24.4T and 94.6 % to Halobellus clavatus TNN18T. The rpoB' gene sequence similarity of strain S2FP14T was 97.4 % to 87.6 % with members of genus Halobellus. The major phospholipids of strain S2FP14T were phosphatidylglycerol phosphate methyl ester and phosphatidylglycerosulfate, plus a very small amount of phosphatidylglycerol and an archaeal analogue of bisphosphatidylglycerol. With regard to glycolipid composition, the most abundant glycolipids were the sulfated diglycosyl diphytanilglyceroldiether and a glycosyl-cardiolipin. The G+C content of strain S2FP14T genomic DNA was 61.4 mol%. The DNA-DNA hybridization between strain S2FP14T and Halobellus inordinatus JCM 18361T was 51 %. Based on the phylogenetic, phenotypic and chemotaxonomic features, a novel species, Halobellus ramosii sp. nov. is proposed. The type strain is S2FP14T ( = CECT 8167T = DSM 26177T).

Delivery systems for natural antioxidant compounds: Archaeosomes and archaeosomal hydrogels characterization and release study.

The aim of this study was to use archaeosomes, a novel kind of liposomes made up by archaeal polar lipids, both multilamellars (MLVs) and unilamellars (SUVs), as a topical delivery system for natural antioxidant compounds recovered from olive mill waste. For comparative purpose an analogue formulation of phosphatidylcholine liposomes was prepared. SUVs were smaller than MLVs ones, showing size values smaller than 200nm, which was maintained during the stability study. Transmission electron microscopy showed spherical morphology for conventional liposomes while archaeosomes had more irregular membranes. Vesicle encapsulation efficiency was quite similar in both formulations and was enough to ensure a good antioxidant activity. Stability studies were performed one month after the preparation of formulations, which showed a high stability with no change in the initial characteristics of the suspensions. Furthermore, the possibility of incorporating the liposomal suspensions in different excipients (Carbopol-940(®) and Pluronic-127(®)) for topical administration was studied. In order to evaluate the release behaviour of the different systems prepared, in vitro diffusion studies were carried out using vertical diffusion Franz cells. In both cases the incorporation of the vesicles into the gels lead in a sustained release for 24h. Archaeosome gels released a similar amount of phenolic compounds regardless the excipient used, while in liposomal gels great release differences were found between carbopol and pluronic gel.

Marinobacter oulmenensis sp. nov., a moderately halophilic bacterium isolated from brine of a salt concentrator.

A Gram-negative, aerobic, moderately halophilic bacterium, designated Set74(T), was isolated from brine of a salt concentrator at Ain Oulmene, Algeria. The strain grew optimally at 37-40 °C, at pH 6.5-7.0 and with 5-7.5 % (w/v) NaCl and used various organic compounds as sole carbon, nitrogen and energy sources. Ubiquinone 9 (Q-9) was the major lipoquinone. The main cellular fatty acids were C16:0, C18:1ω9c, summed feature 7 (ECL 18.846; C19:0 cyclo ω10c and/or C19:1ω6c), C12:0 3-OH, C16:1ω9c, C18:0 and C12:0. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was 57.4 mol%. The 16S rRNA gene sequence analysis indicated that strain Set74(T) was a member of the genus Marinobacter. The closest relatives of strain Set74(T) were Marinobacter santoriniensis NKSG1(T) (97.5 % 16S rRNA gene sequence similarity) and Marinobacter koreensis DD-M3(T) (97.4 %). DNA-DNA relatedness between strain Set74(T) and M. santoriniensis DSM 21262(T) and M. koreensis DSM 17924(T) was 45 and 37 %, respectively. On the basis of the phenotypic, chemotaxonomic and phylogenetic features, a novel species, Marinobacter oulmenensis sp. nov., is proposed. The type strain is Set74(T) ( = CECT 7499(T)  = DSM 22359(T)).

Archaeosomes as carriers for topical delivery of betamethasone dipropionate: in vitro skin permeation study.

A comparative study between archaeosomes, lipid lamellar vesicles made from archaea polar lipids, and conventional phospholipids liposomes was carried out, aiming at evaluating the properties and the potential of archaeosomes as novel colloidal carriers for effective drug delivery to the skin. Betamethasone dipropionate (BMD)-loaded archaeosomes and conventional liposomes were prepared by the thin-lipid film and sonication procedures, using, respectively, archaeal lipids extracted from archaea Halobacterium salinarum and enriched soy phosphatidylcholine. Vesicular formulations were characterized by assessing vesicle size, zeta potential, incorporation efficiency, and morphology. In order to investigate the effect of the incorporation in the two different colloidal carrier systems on the (trans)dermal delivery of BMD, in vitro drug permeation studies through full-thickness pig skin were carried out by using Franz diffusion vertical cells by testing both archaeal and liposomal dispersions. Interestingly, archaeosomes appeared to be the most effective carriers for the model drug, achieveing a major drug penetration and accumulation in the skin strata, especially in the epidermis. This can, presumably, be due to the enhanced archaeosomal bilayer fluidity, as indicated by the rheological studies that provided insight into the viscoelastic properties of all the studied systems. The available data suggest that suitably developed archaeosomes may hold great promise as delivery vehicles for topical applications.

Marinobacter lacisalsi sp. nov., a moderately halophilic bacterium isolated from the saline-wetland wildfowl reserve Fuente de Piedra in southern Spain.

A Gram-negative, non-spore-forming, motile, moderately halophilic, aerobic, rod-shaped bacterium, designated strain FP2.5(T), was isolated from the inland hypersaline lake Fuente de Piedra, a saline-wetland wildfowl reserve located in the province of Málaga in southern Spain. Strain FP2.5(T) was subjected to a polyphasic taxonomic study. It produced colonies with a light-yellow pigment. Strain FP2.5(T) grew at salinities of 3-15 % (w/v) and at temperatures of 20-40 degrees C. The pH range for growth was 5-9. Strain FP2.5(T) was able to utilize various organic acids as sole carbon and energy source. Its major fatty acids were C(16 : 0), C(18 : 1)omega9c and C(16 : 1)omega9c. The DNA G+C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain FP2.5(T) appeared to be a member of the genus Marinobacter and clustered closely with the type strains of Marinobacter segnicrescens, Marinobacter bryozoorum and Marinobacter gudaonensis (levels of 16S rRNA gene sequence similarity of 98.1, 97.4 and 97.2 %, respectively). However, DNA-DNA relatedness between the new isolate and the type strains of its closest related Marinobacter species was low; levels of DNA-DNA relatedness between strain FP2.5(T) and M. segnicrescens LMG 23928(T), M. bryozoorum DSM 15401(T) and M. gudaonensis DSM 18066(T) were 36.3, 32.1 and 24.9 %, respectively. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, strain FP2.5(T) is considered to represent a novel species of the genus Marinobacter, for which the name Marinobacter lacisalsi sp. nov. is proposed. The type strain is FP2.5(T) (=CECT 7297(T)=LMG 24237(T)).