Immobilization of Bradyrhizobium and Azospirillum in alginate matrix for long time of storage maintains cell viability and interaction with peanut.

Immobilizarion of PGPR for agricultural applications aims to provide temporary physical protection from stressful environmental conditions and the gradual release of cells for successful root colonization, release the cells gradually. In this work, we immobilized Bradyrhizobium sp. SEMIA6144 or Azospirillum brasilense Az39 cells in 2% alginate beads prepared by ionic gelation process, and then stored up to 12 months at 4 °C. Alginate matrix showed interaction with the immobilized bacteria (FTIR), allowed a constant release of cells, and improved their viability and capability to interact with Arachis hypogaea. Cell number into beads reached 107 CFU.bead-1; however, viability decreased from 4 months of storage for Az39, while it was maintained up to 12 months for SEMIA6144, showing a low metabolic activity measured by the MTT assay. Adhesion of SEMIA6144 and Az39 from new beads to peanut root was 11.5% and 16%, respectively, higher than non-immobilized bacteria. Peanut inoculation with 12 months storage SEMIA6144 beads significantly increased root length and biomass at 30 days of growth, and under restrictive water condition (RWC), nodulation and total plant N content increased compared with liquid inoculation. Our results demonstrate that immobilization of SEMIA6144 and Az39 in alginate matrix is a potential alternative to enhance peanut growth even under RWC. KEY POINTS: • Alginate encapsulation enhances viability of SEMIA6144 or Az39 under storage at 4 °C for 1 year. • Alginate beads 2% ensure the gradual release of the microorganisms. • Cells from beads stored for long periods present chemotaxis and adhesion to peanut root. • Peanut inoculation with 1-year-old SEMIA6144 beads improves nodulation and growth in RWC.

Development of micropatterning polyimide films for enhanced antifouling and antibacterial properties.

A commercial biomedical Polyimide (PI) film was topographically and chemically modified by generating micrometric periodic arrays of lines using Direct Laser Interference Patterning (DLIP) in order to improve antifouling and antibacterial properties. DLIP patterning was performed with periods from 1 µm to 10 µm. The physical modification of the surface was characterized by SEM, AFM and contact angle measurements and, the chemical composition of the ablated surfaces was analyzed by ATR-IR and XPS spectroscopies. The antibacterial effects were evaluated through the effect on Pseudomonas aeruginosa colonies growth on the LB (Luria Bertani) broth. The results showed that the laser treatment change the topography and as a consequence the chemistry surface, also that the microstructured surfaces with periods below 2 µm, exhibited a significant bacterial (P. aeruginosa) adhesion decrease compared with non-structured surfaces or with surfaces with periods higher than 2 µm. The results suggest that periodic topography only confer antifouling properties and reduction of the biofilm formation when the microstructure presents periods ranging from 1 µm to 2 µm. On the other hand, the topography that confer strong antifouling superficial properties persists at long incubation times. In that way, polymer applications in the biosciences field can be improved by a surface topography modification using a simple, single-step laser-assisted ablation method.

Highly effective antimicrobial nanocomposites based on hydrogel matrix and silver nanoparticles: long-lasting bactericidal and bacteriostatic effects.

Antimicrobial nanocomposites (NCs) are being used as an alternative antibacterial therapy for killing antibiotic-resistant pathogenic bacteria. The NCs are made of Ag nanoparticles (AgNPs) inside biocompatible hydrogel matrixes. The NCs were synthesized by the absorption of AgNO3 solution into a hydrogel matrix, followed by UV light irradiation, without using additional toxic reactants. The hydrogels used as matrixes are based on N-isopropylacrylamide (NIPAM) and copolymers with different functional groups: 2-acrylamide-2-methylpropanesulfonic acid (AMPS), N-hydroxyethylacrylamide (HEAA) and (3-acrylamidepropil)trimethylammonium chloride (APTMAC). Neutral, anionic and cationic groups were added to the matrixes in order to study their effects on the release of antibacterial species. The NCs were characterized by UV-visible spectroscopy and transmission electronic microscopy. The kinetics of the release of Ag+ ions from the NCs were followed by UV-visible spectroscopy at 300 nm. Biological experiments were based on the plate count method and agar diffusion testing against Pseudomonas aeruginosa. The bacterial death rate using the NCs is higher than when PNIPAM and nanoparticles in solution are used and seems to be related to the large amount of AgNPs contained inside the gels. In all cases, inhibition and diffusion halos were observed upon the exposure of bacterial cultures on agar to NC discs. The presence of both halos confirmed the bactericidal and bacteriostatic effects of the NCs. The reusability (prolonged use) of the materials was demonstrated until the Ag-NP content was exhausted. The NCs with a higher antibacterial capacity are based on a PNIPAM-co-6%APTMAC matrix. It was demonstrated that these NC materials have the capacity to maintain an aseptic/antiseptic zone for 7 to 15 days.

Photothermally enhanced bactericidal activity by the combined effect of NIR laser and unmodified graphene oxide against Pseudomonas aeruginosa.

The manuscript shows the application of unmodified graphene oxide (GO) as a photothermally susceptible material to trigger antibacterial effects. The synthesis and characterization of unmodified GO easily dispersed in aqueous solutions is also shown. High GO concentrations in the dark and low GO concentrations irradiated with near infrared light (NIR) produced death in nosocomial bacterium (Pseudomonas aeruginosa). It is demonstrated that GO dispersion in the dark produced a dose-dependent increase in the antibacterial action at concentrations up to 120 µg/mL. On the other hand, by using much lower concentrations (c.a. 2 µg/mL) of GO (non toxic in the dark) and irradiating with near-infrared radiation during 15 min, a degree of mortality of 98.49% was observed. The P. aeruginosa treated with GO and irradiated exhibited DNA fragmentation due to the physical damage of cell membranes. The GO 2 µg/mL dispersions proved favorable, since they do not induce cell death in the dark, whereas the combination with NIR light triggers the damage to the cell membranes. This characteristic is clearly an advantage in comparison with traditional antibacterial nanomaterials (such as nanoparticles), which induce cell killing due to the nanoparticles toxicity per se. Furthermore, this work provides a novel treatment for combating bacterial nosocomial infections without the use of antibiotics, opening a new area of clinical application via simple photothermal therapy.

Synthesis of polyaniline (PANI) and functionalized polyaniline (F-PANI) nanoparticles with controlled size by solvent displacement method. Application in fluorescence detection and bacteria killing by photothermal effect.

Polyaniline nanoparticles (PANI-NPs) were easily obtained applying the solvent displacement method by using N-methylpyrrolidone (NMP) as good solvent and water as poor solvent. Different polymers such as polyvinylpyrrolidone (PVP), chondroitin sulfate (ChS), polyvinyl alcohol (PVA), and polyacrylic acid (PAA) were used as stabilizers. Dynamic light scattering and scanning electron microscopy corroborated the size and morphology of the formed NPs. It was demonstrated that the size of nanoparticles could be controlled by setting the concentration of PANI in NMP, the NMP to water ratio, and the stabilizer's nature. The functionalization and fluorescence of NPs were checked by spectroscopic techniques. Since polyaniline show only weak intrinsic luminescence, fluorescent groups were linked to the polyaniline chains prior to the nanoparticle formation using a linker. Polyaniline chains were functionalized by nucleophilic addition of cysteamine trough the thiol group thereby incorporating pendant primary aliphatic amine groups to the polyaniline backbone. Then, dansyl chloride (DNS-Cl), which could act as an extrinsic chromophore, was conjugated to the amine pendant groups. Later, the functionalized polyaniline was used to produce nanoparticles by solvent displacement. The optical and functional properties of fluorescent nanoparticles (F-PANI-NPs) were determined. F-PANI-NPs in the conductive state (pH < 4) are able to absorb near infrared radiation (NIR) creating a photothermal effect in an aqueous medium. Thus, multifunctional nanoparticles are obtained. The application of NIR on a F-PANI-NPs dispersion in contact with Pseudomonas aeruginosa causes bacterial death. Therefore, the F-PANI-NPs could be tracked and applied to inhibit different diseases caused by pathogenic microorganisms and resistant to antibiotics as well as a new disinfection method to surgical materials.

Hybrid biomaterials based on calcium carbonate and polyaniline nanoparticles for application in photothermal therapy.

Inorganic materials contain remarkable properties for drug delivery, such as a large surface area and nanoporous structure. Among these materials, CaCO3 microparticles (CMPs) exhibit a high encapsulation efficiency and solubility in acidic media. The extracellular pH of tumor neoplastic tissue is significantly lower than the extracellular pH of normal tissue facilitating the release of drug-encapsulating CMPs in this area. Conducting polyaniline (PANI) absorbs light energy and transforms it into localized heat to produce cell death. This work aimed to generate hybrid CMPs loaded with PANI for photothermal therapy (PTT). The hybrid nanomaterial was synthesized with CaCO3 and carboxymethyl cellulose in a simple, reproducible manner. The CMP-PANI-Cys particles were developed for the first time and represent a novel type of hybrid biomaterial. Resultant nanoparticles were characterized utilizing scanning electron microscopy, dynamic light scattering, zeta potential, UV-vis, FTIR and Raman spectroscopy. In vitro HeLa cells in dark and irradiated conditions showed that CMP-PANI-Cys and PANI-Cys are nontoxic at the assayed concentrations. Hybrid biomaterials displayed high efficiency for potential PTT compared with PANI-Cys. In summary, hierarchical hybrid biomaterials composed of CMPs and PANI-Cys combined with near infrared irradiation represents a useful alternative in PTT.

Cysteine modified polyaniline films improve biocompatibility for two cell lines.

This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering.

Assessment of polyaniline nanoparticles toxicity and teratogenicity in aquatic environment using Rhinella arenarum model.

With the rapid growth of nanotechnology and the applications of nanoparticles, environmental exposure to these particles is increasing. However, their impact in human and environmental health is not well studied. Anurans, with life stage comprising embryos, tadpoles and adults, have an extremely permeable skin which makes them excellent indicators of environmental health. This study evaluated the acute toxicity effects of polyaniline nanoparticles (PANI-Np) in different dispersant on embryos and larvae of Rhinella arenarum. The results showed that LC50 of PANI-Np dispersed in polyvinylpyrrolidone (PVP) were 1,500 mg/L, while LC50 by PANI-Np dispersed in PVP+PNIPAM (polyN-isopropylacrilamide) showed a highest toxicity (1,170 mg/L). The embryo teratogenicity increased with increasing exposure concentration in both kinds of PANI-Np although in PANI-Np1, there is an increased teratogenic effect associated with the polymer stabilizer PVP.

Polyaniline nanofibers: acute toxicity and teratogenic effect on Rhinella arenarum embryos.

The fate and effect of nanomaterials in the environment is of paramount importance towards the technological application of the materials. This work shows the ecotoxicological potential of polyaniline (PANI) nanofibers in the larvae Rhinella arenarum by means of AMPHITOX test. Acute toxicity of PANI nanofibers towards embryos of the common South American toad R. arenarum (Anura: bufonidae) was evaluated in the premetamorphosis (stage 25) larvae. The exposure of R. arenarum larvae to at dose of 150, 250 and 400 mg L(-1) resulted in 100% viability within 96 h exposure. The embryos at 2-4 blastomers stage (early life stage teratogenic test) revealed that embryos were not killed and no teratogenic effects were observed when embryos were incubated with PANI nanofibers (150 and 250 mg L(-1)), while only a growth retardation of embryos was induced at levels of 250 mg PANI nanofibers L(-1). On the other hand, at 400 mg L(-1) concentration, a reduction in the body length of larvae and tail malformation was observed. This results suggest that a concentration-dependent toxicity is operative, typified by phenotypes that had abnormal body axes. The presence of PANI nanofibers in gut contents and its excretion by larval stages of R. arenarum was confirmed by UV-visible spectroscopy.

Comparative photodynamic therapy study using two phthalocyanine derivatives.

In the present study, a comparative photodynamic therapy (PDT) study was performed using the phthalocyanine derivatives, ZnPc(OCH(3))(4) and ZnPc(CF(3))(4), in a mouse tumor model, under identical experimental procedures. We studied the ablation of tumors induced by PDT. The end-point was to compare the photodynamic efficacy of ZnPc(OCH(3))(4) and ZnPc(CF(3))(4). ZnPc(OCH(3))(4) and ZnPc(CF(3))(4) were administered intraperitoneally at a dose of 0.2 mg/kg body weight. The injections of drugs were carried out in Balb/c mice bearing subcutaneously inoculated LM2 mouse mammary adenocarcinoma. Histological examination and serum biochemical parameters were used to evaluate hepatic and renal toxicity and function. Phototherapeutic studies were achieved employing a light intensity of 210 J/cm(2). After PDT, tumoral regression analyses were carried out, and the degree of tumor cell death was measured utilizing the vital stain Evan's blue. In this pilot study, we revealed that the cytotoxic effect of ZnPc(OCH(3))(4) after PDT led to a higher success rate compared to ZnPc(CF(3))(4)-PDT when both were intraperitoneally injectioned. Both phthalocynanine derivatives were able to induce ablation in the tumors. In summary, these results demonstrate the feasibility of ZnPc(OCH(3))(4)- or ZnPc(CF(3))(4)-PDT and its potential as a treatment for small tumors.

Easy way to fabricate nanostructures on a reactive polymer surface.

The fabrication of advanced architectures in poly(glycidylmethacrylate-co-styrene) (PGMA-S) copolymers using direct laser interference patterning (DLIP) and its selective functionalization is reported. The structure features depend mainly on the laser energy used and on the styrene content in the copolymer. The topography, measured by electronic scanning microscopy, show regular and ordered arrays for the polystyrene (PS) and for the copolymers PGMA-S. The surface PS homopolymer is ablated at the position of maximum light fluence (constructive interference), while in the copolymers the surfaces swell up at the regions with maximal fluence. The styrene units are shown to absorb the laser energy giving photothermally ablated regions or promoting the chemical decomposition of acrylate units or polymer segments. In that way, DLIP provides a unique way to produce regularly ordered structures protruding or depressing from the polymer surface without altering to a large extent the chemical nature of the material. In addition, it is shown, using fluorescence microscopy, that amine-polyethylenglycol-CdSe quantum dots (NH(2)-PEG-QDs) could be spatially localized by reaction with patterned surfaces of PGMA-S. In that way, it is proven that a patterned and chemically reactive surface can be created using DLIP of PGMA-S.

Pharmacokinetic, toxicological and phototherapeutic studies of phthalocyanine ZnPcCF3.

Photodynamic therapy (PDT) is an alternative modality for cancer therapy. It induces neoplasic cells death through photoachievable sensitizers. The aim of this work was to evaluate the pharmacokinetic, toxic and phototherapeutic effects of the phthalocyanine ZnPcCF(3) in a Balb/c mice tumor model. Biodistribution studies were carried out by intraperitoneal injection of 0.2mg/kg ZnPcCF(3). Histological studies and serum biochemical parameters were used to evaluate hepatic and renal toxicity and functionality. After tumor irradiation (210J/cm(2)), an analysis of tumor necrosis degree was used to evaluate the phototherapeutic effects. It was measured at 1, 2, 3 and 4 days after PDT. Vital staining was performed by intraperitoneal injection of 0.35ml 1% Evans Blue solution. Six hours later, tumors were excised and examined. The unstained area was attributed to necrotic tissue, whereas the stained area showed tissue with preserved blood supply. ZnPcCF(3) was accumulated in spleen, liver and duodenum. It suggests that ZnPcCF(3) is eliminated from the body via bile-gut. The phthalocyanine was not found in brain, therefore, it would not cross the blood-brain barrier, thus toxicity risk in the central nervous system is not probable. Moreover, ZnPcCF(3) does not accumulate in skin, it would eliminate cutaneous photosensitizing risks. The dose of 0.2mg/kg ZnPcCF(3) resulted in a low acute toxicity with revertible damages, which indicates that this dose can be used for PDT. The tumor death was of 89% 4 days after PDT. It indicates that ZnPcCF(3) would be effective in PDT.

Zinc-(II) 2,9,16,23-tetrakis (methoxy) phthalocyanine: potential photosensitizer for use in photodynamic therapy in vitro.

Photodynamic therapy (PDT) is an innovative treatment for several types of malignant and non-malignant disease. In the present study, ZnPcOCH(3) was investigated on a human larynx-carcinoma cell line (Hep-2) for its use in PDT. This drug exhibited favourable properties as a photosensitizer in vitro because ZnPcOCH(3) is able to penetrate efficiently in the cytoplasm of cultured cancer cells and is partially localized in lysosomes. The results show that ZnPcOCH(3)-PDT-induced apoptosis by caspase dependent pathway. The new compound shows a good photosensitizing efficiency in vitro on Hep-2 cells, encouraging further in vivo studies.