Antibiofilm Assays Research Articles

Synthesis, Characterization, and Biological Activity Evaluation of Magnetite-Functionalized Eugenol

This work reports the magnetite-functionalization and biological evaluation of eugenol by the co-precipitation method employed only Fe2+ under mild conditions and control from the amount of the incorporated magnetite. Magnetic nanoparticles were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), hydrodynamic size distribution (Zetasizer), and vibrating sample magnetometer (VSM). SEM images showed that EUG·Fe3O4 similar in shape to a nanoflower. The FTIR spectrum confirmed the presence of characteristic EUG and Fe3O4 bands in the EUG·Fe3O4 sample, the XRD analysis showed that the magnetite functionalization with eugenol slightly affected the Fe3O4 crystal structure, while the VSM measurements demonstrate that EUG·Fe3O4 1:1 shows a superparamagnetic behavior, suggesting small non-interacting particles. The in vitro safety profile and cytotoxicity of free eugenol, magnetite pristine, EUG·Fe3O4 1:1, EUG·Fe3O4 1:5, and EUG·Fe3O4 1:10 was investigated using human cell lines (keratinocytes and melanoma). The results demonstrate the high biocompatibility of EUG·Fe3O4 in HaCat cells and the greater specificity for the A375 cell line. Furthermore, the magnetite-functionalization with eugenol decreased the toxic effects of free eugenol on healthy cells. Antibacterial tests were performed in different bacterial strains. The experimental data showed that among the magnetic compounds, the microorganisms were only sensitive to treatment with EUG·Fe3O4 1:1. Regarding the antibiofilm activity assay, it can be observed that only the EUG·Fe3O4 caused a significant decrease in biomass when compared to the positive control. Finally, it can be concluded that EUG·Fe3O4 proves to be a potential candidate for future studies for drug delivery of cancer and bacterial infections treatments.

Antibacterial and Anti-biofilm- Forming Activity of Secondary Metabolites from Sansevieria trifasciata- Leaves Against Pseudomonas aeruginosa

Sansevieria trifasciata is a herbaceous plant frequently used in traditional medicine with antibacterial potency against Pseudomonas aeruginosa. P. aeruginosa is a known bacterial pathogen that often becomes resistant to traditional antibiotics due to its ability to form biofilms. The objective of this research was to determine whether the ethanolic extract of S. trifasciata has any antibacterial or anti-biofilm-forming activity against P. aeruginosa. Bacterial cell damage with a potential fraction of S. trifasciata ethanolic extract treatment was observed by scanning electron microscopy (SEM). An Anti-biofilm assay was perfomed in 96 wells with crystal violet stain. The minimum inhibitory concentration (MIC) results revealed that at a concentration of 4 mg/mL, the potential fraction of S. trifasciata ethanolic could inhibit bacterial growth. At this concentration, the potential fraction of S. trifasciata ethanolic extract could inhibit the biofilm by 60%. The identification of compounds using gas chromatography-Mass Spectrometry (GC-MS) revealed that Neophytadiene was the main active component in the extract. These compounds have the potential to be used as therapeutic agents in the prevention of bacterial biofilms and virulence-related infectious illnesses

Betaine-urea deep eutectic solvent improves imipenem antibiotic activity

Beta-lactam antibiotics are highly unstable in aqueous media, which may lead to subclinical concentrations, antimicrobial resistance and therapeutic failure. In previous work we demonstrated that a natural deep eutectic solvent consisting of betaine and urea (BU) is capable of improving the stability of some beta-lactams, including imipenem (IMP), the most unstable antibiotic of the family. Here, IMP-BU was studied by selective protonic Nuclear Overhauser Effect Spectroscopy Magnetic Resonance (H1 NOESY NMR) to gain insight into the mechanism by which BU protects IMP. The kinetics of IMP release and its antibacterial activity were evaluated in diffusional, time-kill and antibiofilm assays. It was found that BU is a protective matrix which allows a fast release of IMP, resulting in superior antibacterial activity when compared to IMP in aqueous solution, both against bacteria growing in planktonic form and in biofilms. Furthermore, it was shown that BU is nontoxic when evaluated in fibroblast primary cell cultures and in organotypic skin cultures, and is not immunogenic when tested in vitro in macrophage cultures, suggesting that BU has potential application as a biomaterial or excipient.

Dual-sensitive antibacterial peptide nanoparticles prevent dental caries.

Background: Dental caries is the most prevalent bacterial biofilm-induced disease. Current clinical prevention and treatment agents often suffer from adverse effects on oral microbiota diversity and normal tissues, predominately arising from the poor biofilm-targeting property of the agents.Methods: To address this concern, we herein report dual-sensitive antibacterial peptide nanoparticles pHly-1 NPs upon acid and lipid-binding for treatment of dental caries. Amino acid substitutions were performed to design the peptide pHly-1. The potential, morphology and secondary structure of pHly-1 were characterized to elucidate the mechanisms of its pH and lipid sensitivity. Bacterial membrane integrity assay and RNA-seq were applied to uncover the antimicrobial mechanism of peptides under acidic condition. The in vitro and ex vivo antibiofilm assays were used to determine the antibiofilm performance of pHly-1 NPs. We also carried out the in vivo anti-caries treatment by pHly-1 NPs on dental caries animal model. Oral microbiome and histopathological analyses were performed to assess the in vivo safety of pHly-1 NPs.Results: The pHly-1 peptide underwent the coil-helix conformational transition upon binding to bacterial membranes in the acidic cariogenic biofilm microenvironment, thereby killing cariogenic bacteria. Under normal physiological conditions, pHly-1 adopted a β-sheet conformation and formed nanofibers, resulting in negligible cytotoxicity towards oral microbes. However, in acidic solution, pHly-1 NPs displayed reliable antibacterial activity against Streptococcus mutans, including standard and clinically isolated strains, mainly via cell membrane disruption, and also suppressed in vitro and human-derived ex vivo biofilm development. Compared to the clinical agent chlorhexidine, in vivo topical treatment with pHly-1 NPs showed an advanced effect on inhibiting rat dental caries development without adverse effects on oral microbiota diversity and normal oral or gastric tissues.Conclusion: Our results demonstrated the high efficacy of dual-sensitive antimicrobial peptides for the selective damage of bacterial biofilms, providing an efficient strategy for preventing and treating dental caries.

Evaluation of the antibiofilm and antimicrobial activities of Glycyrrhiza glabra and Viola odorata in poly herbal formulations

Traditional polyherbal formulations are essential part of health system in various populations in the subcontinent region including Pakistan, however scientific evaluation of such claims are not significant. In this investigation, the antibiofilm and antimicrobial activities of Glycyrrhiza glabra and Viola odorata, two important ingredients of a traditional herbal formulation were determined in synergism. Various fractions of plants extract were prepared in the order of polarity. All fractions were tested against Gram negative (Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus and Salmonella typhi) bacteria and antimicrobial activity was assessed using agar diffusion method. The MIC (Minimum Inhibitory concentration), was assessed. Finally antibiofilm activities were determined using 96 micro wells plate by calculating percent inhibition. In case of Viola oldorata, the inhibition zone ranged between (6 mm to 18 mm±1.24) against the test strains. The highest inhibition was recorded against gram positive bacteria Strep: Pneumoniae (20 mm±0.99) followed by S. aureus (18 mm±1.02) using methanolic extract. A moderate level of inhibition was seen in case of Glycyrrhiza glabra against the microbial strains. It was noticed that Glycyrrhiza Glabra was more active against gram negative bacteria. The methanolic fraction presented highest activity against e coli (18 mm±0.96) followed by K. pneumoniae and P. aureginosa (12 mm±1.22).The MIC ranged between 4–8 mg/mL. During antibiofilm assay, a moderate level of inhibition occurs using the Viola odorata (40%inhibition) and Glycyrrhiza glabra (45%inhibition), that increased by using combination of both plants in 50:50 ratio. It was concluded that the effective properties of indigenous poly herbal formulation are due to synergism causing greater antibiofilm characteristics against the biofilm producer strains as well as other pathogenic microorganisms.

A nano phototheranostic approach of toluidine blue conjugated gold silver core shells mediated photodynamic therapy to treat diabetic foot ulcer

Diabetic foot infection caused by multidrug-resistant bacteria, is becoming serious problem. Moreover, polymicrobial biofilms contribute significantly to the persistent infections. In the present study, we investigated the effectiveness of novel toluidine blue conjugated chitosan coated gold–silver core–shell nanoparticles (TBO–chit–Au–AgNPs) mediated photodynamic therapy and demonstrate their use as a nontoxic antibacterial therapy to combat diabetic foot ulcer (DFU) caused by multi-drug resistant strains both in monomicrobial and polymicrobial state of infection. In vitro efficacy of TBO–chit–Au–AgNPs mediated photodynamic therapy (PDT) against polymicrobial biofilms was determined using standard plate count method and compared with that of monomicrobial biofilms of each species. Different anti-biofilm assays and microscopic studies were performed to check the efficacy of TBO–chit–Au–AgNPs mediated PDT, displayed significant decrease in the formation of biofilm. Finally, its therapeutic potential was validated in vivo type-2DFU. Cytokines level was found reduced, using nano-phototheranostic approach, indicating infection control. Expression profile of growth factors confirmed both the pathogenesis and healing of DFU. Hence, we conclude that TBO–chit–Au–AgNPs mediated PDT is a promising anti-bacterial therapeutic approach which leads to a synergistic healing of DFU caused by MDR bacterial strains.

Biologically formed silver nanoparticles and in vitro study of their antimicrobial activities on resistant pathogens.

Background and Objectives:Silver nanoparticles (AgNPs) have been found to have multiple uses as antibacterial, antifungal and anti-biofilm agents because of their biological activities and safety. The present study was aimed to analyze the antimicrobial and anti-biofilm activities as well as the cytotoxic effect of AgNPs against different human pathogens.Materials and Methods:AgNPs were synthesized using cell free supernatants of Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 19433), Pseudomonas aeruginosa (ATCC 27856), Enterobacter cloacae (ATCC 13047) and Penicillium oxalicum strain, then were analyzed using UV/Vis Spectral Analysis, Transmission electron microscopy (TEM). Scanning Electron Microscope (SEM) and Energy Dispersive-X-ray Spectroscopy (EDX) analysis. Antimicrobial activities of biosynthesized AgNPs were assessed with selected antimicrobial agents against multidrug resistant bacteria and candida. Anti-biofilm and cytotoxicity assays of these biosynthesized AgNPs were also done.Results:The synthesis of AgNPs were confirmed through observed color change and monitoring UV-Vis spectrum which showed homogeneous (little agglomeration) distribution of silver nanoparticles. TEM and SEM have shown that the particle size ranged from 13 to 34 (nm) with spherical shape and a high signal with EDX analysis. Antibacterial and antifungal efficacy of antibiotics and fluconazole were increased in combination with biosynthesized AgNPs against resistant bacteria and candida. Significant reduction in biofilm formation was found better with Penicillium oxalicum AgNPs against biofilm forming bacteria.Conclusion:Penicillium oxalicum has the best effect towards synthesizing AgNPs, for antimicrobial activities against resistant bacteria and candida, in addition to anti-biofilm activities against biofilm forming Staphylococcus aureus and E. coli and the safest cytotoxicity effect on (MRC-5) cell line.

Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro.

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3’hydroxychalcone (3’chalc) -loaded nanoemulsion (NE) (NE3’chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3’chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3’hidroxychalcone (3’chalc) range from 0.97- 7.8 µg/mL and NE3’chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3’chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3’chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3’chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

Bacterial Protease Inhibitors as Antibacterial agents to prevent Bacterial Infections Associated with Biofilms

Isolation of protease inhibitor producing bacteria from microbial mat and investigating its anti-biofilm potential against biofilm producing organism was selected as the main objective of the present study. Protease inhibitor (PI) was produced from bacterial isolates and purified using ammonium sulphate precipitation methods. Primary and secondary protease inhibitor assay was carried out separately to confirm the inhibition of protease enzyme activity both qualitatively and quantitatively. Antibacterial activity and anti-biofilm assay was performed to determine the biofilm prevention capabilities of PI. Three isolates (B1PI, B2PI and B3PI) were screened and B2PI bacterial culture was selected based on the results of primary and secondary protease inhibitor assay. Maximum trypsin inhibition of 77.5±0.25% was recorded for the isolate B2PI. Antibacterial activity of the B2PI protease inhibitor fractions exhibited inhibitory zones of 22.3±1.04mm and 20.2±0.25mm against Escherichia coli and Staphylococcus aureus respectively. Anti-biofilm assay of protease inhibitor fractions expressed 31.2μl/ml of MBIC against Escherichia coli and Staphylococcus aureus. The results conclude that, the protease inhibitor from the microbial mat isolate will be an effective alternative to the commercial antibiotics either alone or in combination with other drugs synergistically which shall be studied elaborately in future.

Antimicrobial photodynamic therapy (aPDT) against vancomycin resistant Staphylococcus aureus (VRSA) biofilm disruption: A putative role of phagocytosis in infection control

Biofilm mediated infections have major clinical impact. Staphylococcus aureus is a pathogen that frequently causes biofilm forming infections, such as those associated with medical devices and persistent wounds. Microorganisms embedded in biofilm are impervious to antibiotics and other antimicrobial agents, thus they are difficult to eliminate. The upsurge of multi-drug resistant strains makes treating such illnesses even more difficult. Therefore, new strategies are required to combat such type of infections. In this work, we have proposed an alternative therapeutic option to eradicate preformed biofilm of vancomycin resistant Staphylococcus aureus (VRSA) and enhanced phagocytosis by neutrophils in fresh human blood using curcumin mediated antimicrobial photodynamic therapy (aPDT).At sub-MIC of curcumin, different anti-biofilm assays and microscopic examinations were performed, followed by 20 J/cm2 of blue laser light irradiation which corresponds to 52 s only. The result showed significant disruption of VRSA biofilm. Moreover, when curcumin-aPDT treated VRSA biofilm was exposed to whole blood from healthy donors, it was nearly completely eradicated. The present study suggests that curcumin-aPDT enhanced phagocytosis may be a useful strategy for inactivating VRSA biofilms adhering to medical implant surfaces.

Positively-charged microcrystalline cellulose microparticles: Rapid killing effect on bacteria, trapping behavior and excellent elimination efficiency of biofilm matrix from water environment

Pathogen and biofilm contamination in aqueous systems leave millions of people at risk of waterborne diseases. Herein, to address this issue, a green and highly efficient strategy is developed to concurrently trap and kill bacteria, eliminate the debris and the existing biofilm matrix in water environment via magnetic microparticles. The particles (TPFPs) were prepared from the in-situ deposition of Fe3O4 nanoparticles onto the surface of antibacterial functionalized microcrystalline cellulose (MCC). Noticeably, TPFPs can completely inactivate both S. aureus and E. coli once contacting for 30 min by disrupting the bacterial membrane. Meanwhile, the MCC-based magnetic particles retained 100% biocidal efficiency against E. coli (5 * 104E. coli/mg particles) during ten recycling procedures without any treatment. More importantly, according to the results of trapping behavior and antibiofilm assays, not only bacteria could be captured by the particles (trapping rate was over 85%), but also the residual debris from dead bacteria and fragmented biofilm was together removed based on the special structure and functions of the antibacterial particles (~ 80%), including extremely rough surfaces, surficial positive charge and magneto-responsive property. This study presents an efficient approach for microorganism management in water system which can be expectantly applied to improve the water safety

Pectin-cellulose hydrogel, silk fibroin and magnesium hydroxide nanoparticles hybrid nanocomposites for biomedical applications

Natural polymers are at the center of materials development for biomedical and biotechnological applications based on their biocompatibility, low-toxicity and biodegradability. In this study, a novel nanobiocomposite based on cross-linked pectin-cellulose hydrogel, silk fibroin, and Mg(OH)2 nanoparticles was designed and synthesized. After extensive physical-chemical characterization, the biological response of pectin-cellulose/silk fibroin/Mg(OH)2 nanobiocomposite scaffolds was evaluated by cell viability, red blood cells hemolytic and anti-biofilm assays. After 3 days and 7 days, the cell viability of this nanobiocomposite scaffold was 65.5% and 60.5% respectively. The hemolytic effect was below 20%. Furthermore, the presence of silk fibroin and Mg(OH)2 nanoparticles allowed to enhance the anti-biofilm activity, inhibiting the P. aeruginosa biofilm formation.

Pillar[5]arene-Based Polycationic Glyco[2]rotaxanes Designed as Pseudomonas aeruginosa Antibiofilm Agents.

Pseudomonas aeruginosa (P.A.) is a human pathogen belonging to the top priorities for the discovery of new therapeutic solutions. Its propensity to generate biofilms strongly complicates the treatments required to cure P.A. infections. Herein, we describe the synthesis of a series of novel rotaxanes composed of a central galactosylated pillar[5]arene, a tetrafucosylated dendron, and a tetraguanidinium subunit. Besides the high affinity of the final glycorotaxanes for the two P.A. lectins LecA and LecB, potent inhibition levels of biofilm growth were evidenced, showing that their three subunits work synergistically. An antibiofilm assay using a double ΔlecAΔlecB mutant compared to the wild type demonstrated that the antibiofilm activity of the best glycorotaxane is lectin-mediated. Such antibiofilm potency had rarely been reached in the literature. Importantly, none of the final rotaxanes was bactericidal, showing that their antibiofilm activity does not depend on bacteria killing, which is a rare feature for antibiofilm agents.

Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity.

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

Antifungal Activity of Extracts, Fractions, and Constituents from Coccoloba cowellii Leaves.

Coccoloba cowellii Britton (Polygonaceae, order Caryophyllales) is an endemic and critically endangered plant species that only grows in the municipality of Camagüey, a province of Cuba. A preliminary investigation of its total methanolic extract led to the discovery of promising antifungal activity. In this study, a bioassay-guided fractionation allowed the isolation of quercetin and four methoxyflavonoids: 3-O-methylquercetin, myricetin 3,3′,4′-trimethyl ether, 6-methoxymyricetin 3,4′-dimethyl ether, and 6-methoxymyricetin 3,3′,4′-trimethyl ether. The leaf extract, fractions, and compounds were tested against various fungi and showed strong in vitro antifungal activity against Cryptococcus neoformans and various Candida spp. with no cytotoxicity (CC50 > 64.0 µg/mL) on MRC-5 SV2 cells, determined by a resazurin assay. A Candida albicans SC5314 antibiofilm assay indicated that the antifungal activity of C. cowellii extracts and constituents is mainly targeted to planktonic cells. The total methanolic extract showed higher and broader activity compared with the fractions and mixture of compounds.

Spilanthol content of Acmella oleracea subtypes and their bactericide and antibiofilm activities against Streptococcus mutans

This study aimed to determine the extractive yield, the spilanthol content and the bactericide and antibiofilm activities of aqueous and hydroethanolic extracts of two regional variations (Amapá and Pará) of Acmella oleracea (L.) R. K. Jansen against the cariogenic bacteria Streptococcus mutans. Aqueous extracts of leaves and hydroethanolic extracts of leaves and stems were obtained and submitted to UHPLC-ESI-QToF-MS/MS characterization. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts were determined using the microdilution method, while the crystal violet assay was used to access the activity over S. mutans pre-formed 1-day biofilms. The aqueous extracts of the leaves showed higher extractive yields compared to the hydroethanolic solutions. Nonetheless, the hydroethanolic mixture ensured a higher extraction of spilanthol. For instance, the leaves of Pará variation (EELAO-PA) (99.97%) and the stem of Amapá variation (EESAO-AP) (99.17%) presented the highest spilanthol content. Therefore, these extracts were selected for the antimicrobial assays, presenting equally MIC and MBC of 125 µg/ml against S. mutans. In the antibiofilm assay, EELAO-PA at 500 µg/ml (4xMIC) presented an inhibition of 50.89% after a 1-minute single application, similarly (p>0.05) to that found after chlorhexidine gluconate 0.12% treatment. Accordingly, A. oleracea rich spilanthol extracts showed remarkable antibacterial activity against the cariogenic S. mutans and could become an affordable coadjuvant used in dental products to prevent and control dental caries.

Combining topography and peptide to inhibit algae attachment: Preparation of peptide‐modified microstructured surfaces

Marine facilities and ships are easily plagued by biofouling. Synergistic surfaces combining surface reactive ion etching and peptide modification were designed and prepared to tackle surface biofouling. Six microstructural surfaces were first designed and machined based on the contact point theory; the synergistic surfaces were then prepared by peptide modification. Laser confocal scanning microscope (LCSM), scanning electron microscopy (SEM), contact angle measurement, and X‐ray photoelectron spectroscopy (XPS) were utilized to analyze the surface morphology and surface chemical properties; the results demonstrated that six synergistic surfaces were prepared successfully with 0.8‐μm depth, surface contact angle increased from 75° to 116.99°, and the surface chemical compositions were also changed significantly due to peptide modification. Antibiofilm assays showed that surface modification and surface topology could slightly reduce the formation of biofilm. While the synergistic surfaces possess strong anti‐algal performance, and anti‐Chlorella pyrenoidosa (C. pyrenoidosa) and anti‐Phaeodactylum tricornutum (P. tricornutum) rates reached 78.56% and 87.80%, respectively after 7‐day immersion in artificial seawater. Compared with the single surface treatment method, the antifouling performance of the synergetic surfaces were stronger. This strategy demonstrated a realistic paradigm for the further improving surface antifouling performance in a green method.

Concentrated MTA Repair HP reduced biofilm and can cause reparative action at a distance.

To evaluate in vitro whether MTA Repair HP can induce repair processes at a distance, including its effects on biofilm, cell viability, migration, production of TGF-β, phosphate and ALP, evaluated through MTA diluted extracts. Initially, antibacterial tests were performed with the bacterium Streptococcus mutans (ATCC 25175) in the presence of MTA extracts (dilutions of 1:1, 1:2 and 1:4). Growth inhibition assay by microdilution in broth, antibiofilm plate assay of young biofilm and antibiofilm assay in confocal microscopy of mature biofilm were carried out. Then, pulp cells were stimulated in the presence of several MTA dilutions, and cell viability (MTT assay), proliferation and migration capacity (scratch assay) were evaluated. To evaluate the capacity of 1:1, 1:2 and 1:4 dilutions of MTA Repair HP to promote the production of important agents of odontogenic differentiation and mineralization, ALP activity, TGF-β secretion and phosphate quantification were measured. Statistical differences were verified using one-way and two-way anova and Tukey's post-tests. The test dilutions of MTA Repair HP did not inhibit planktonic S.mutans growth but were able to reduce young and mature S.mutans biofilm (p<0.001). In addition, none of the MTA Repair HP dilutions was cytotoxic for pulp cells. The 1:2 and 1:4 dilutions of MTA Repair HP induced migration and proliferation of pulp cells (p<0.05). ALP activity and TGF-β secretion were independent of the tested dilution (p<0.001). Diluted 1:4 MTA Repair HP produced less phosphate than the more concentrated 1:1 and 1:2 MTA dilutions (p<0.001). Undiluted MTA Repair HP reduced S.mutans biofilm, when compared to 1:2 and 1:4 MTA dilutions. Furthermore, none of the tested dilutions was cytotoxic to pulp cells. MTA Repair HP promoted cell migration and proliferation at a distance, assessed through the dilution of the MTA. Even from a distance, MTA Repair HP has the ability to participate in some events related to repair, such as migration, proliferation and TGF production.

Hybrid Bionanocomposite Containing Magnesium Hydroxide Nanoparticles Embedded in a Carboxymethyl Cellulose Hydrogel Plus Silk Fibroin as a Scaffold for Wound Dressing Applications.

Based on the promising biomedical developments in wound healing strategies, herein, a new nanobiocomposite scaffold was designed and presented by incorporation of carboxymethyl cellulose hydrogels prepared using epichlorohydrin as a cross-linking agent (CMC hydrogel), a natural silk fibroin (SF) protein, and magnesium hydroxide nanoparticles (Mg(OH)2 NPs). Biological evaluation of the CMC hydrogel/SF/Mg(OH)2 nanobiocomposite scaffold was conducted via in vitro cell viability assays and in vivo assays, red blood cell hemolysis, and antibiofilm assays. Considering the cell viability percentage of Hu02 cells (84.5%) in the presence of the prepared nanobiocomposite after 7 days, it was indicated that this new nanoscaffold was biocompatible. The signs of excellent hemocompatibility and the high antibacterial activity were observed due to the low-point hemolytic effect (8.3%) and high-level potential in constraining the P. aeruginosa biofilm formation with a low OD value (0.13). Moreover, in vivo wound healing assay results indicated that the wound healing method was faster in mice treated with the prepared nanobiocomposite scaffold (82.29%) than the control group (75.63%) in 12 days. Apart from the structural characterization of the CMC hydrogel/SF/Mg(OH)2 nanobiocomposite through FTIR, EDX, FESEM, and TG analyses, compressive mechanical tests, contact angle, porosity, and swelling ratio studies indicated that the combination of the CMC hydrogel structure with SF protein and Mg(OH)2 NPs could significantly impact Young's modulus (from 11.34 to 10.14 MPa), tensile strength (from 299.35 to 250.78 MPa), elongation at break (12.52 to 12.84%), hydrophilicity, and water uptake capacity (92.5%).

Design and characterization of ellagic acid-loaded zein nanoparticles and their effect on the antioxidant and antibacterial activities

Ellagic acid (EA) has demonstrated remarkable antioxidant and antimicrobial activities. The corn protein zein is a natural biopolymer whose biological and functional properties encourage its use to improve therapeutic nano drug delivery systems. In this study, we aimed to design and characterize EA-loaded zein nanoparticles and to evaluate the impact of nanoencapsulation in the antioxidant and antibacterial properties. The nanoparticles were produced by nanoprecipitation and evaluated in terms of morphology, size, polydispersity, zeta potential, encapsulation efficiency and drug loading. The chemical interactions between EA and zein were studied by FTIR, 1H STD-NMR and Fluorescence spectroscopy. Antioxidant activity was quantified using the DPPH and ABTS radical scavenging methods, while antimicrobial activity was determined in terms of inhibitory, bactericide and antibiofilm assays against Staphylococcus aureus and Pseudomonas aeruginosa. EA-loaded zein nanoparticles were spherical, non-aggregated, below 370 nm and positively charged. Selected nanoparticles showed relevant inhibitory and bactericide activity against S. aureus and P. aeruginosa (MIC < 72 µg/mL). The antioxidant activity (IC50) for the nanoencapsulated EA were 56.02 µg/mL and 136.6 µg/mL in the DPPH and ABTS assays, respectively. Furthermore, EA nanoparticles were found to be efficient in reducing S. aureus and P. aeruginosa biofilms by 60 and 30%, respectively. The 1H STD-NMR, FTIR and Fluorescence combined analyses confirmed the interaction between EA and zein, which could explain the enhancement found in the biological activities studied, as the active phenolic groups of this molecule are likely to become more accessible after the entrapment of EA in the formed zein nanoparticles. From this we can conclude that nanoencapsulation of EA boosted its antioxidant and antibacterial activities; this offers a potential strategy against free radicals as well as preventive and treatment option for infections related to Gram-positive and Gram-negative bacteria.