Porous Nanogold/Polyurethane Scaffolds with Improved Antibiofilm, Mechanical, and Thermal Properties and with Reduced Effects on Cell Viability: A Suitable Material for Soft Tissue Applications.

The use of implants carries on a series of problems, among them infections, poor biocompatibility, high levels of cytotoxicity, and significant mechanical differences between implants and host organs that promote stress shielding effects. These problems indicate that the materials used to make implants must meet essential requirements and high standards for implantations to be successful. In this work, we present the synthesis, characterization and evaluation of the antibiofilm, mechanical, and thermal properties, and cytotoxic effect of a nanocomposite-based scaffold on polyurethane (PU) and gold nanoparticles (AuNPs) for soft tissue applications. The effect of the quantity of AuNPs on the antibacterial activity of nanocomposite scaffolds was evaluated against Staphylococcus epidermidis and Klebsiella spp., with a resulting 99.99% inhibition of both bacteria using a small quantity of nanoparticles. Cytotoxicity was evaluated with the T10 1/2 test against fibroblast cells. The results demonstrated that porous nanogold/PU scaffolds have no toxic effects on fibroblast cells to the 5 day exposition. With respect to mechanical properties, stress-strain curves showed that the compressive modulus and yield strength of PU scaffolds were significantly enhanced by AuNPs (by at least 10 times). This is due to changes in the arrangement of hard segments of PU, which increase the stiffness of the polymer. Thermogravimetric analysis showed that the degradation onset temperature rises with an increase in the quantity of AuNPs. These properties and characteristics demonstrate that porous nanogold/PU scaffolds are suitable material for use in soft tissue implants.

CLPFFD-PEG functionalized NIR-absorbing hollow gold nanospheres and gold nanorods inhibit ß-amyloid aggregation.

Gold nanoparticles with specific optical properties in combination with the CLPFFD peptide that exhibits selectivity for ß-amyloid (Aß) aggregates are promising photothermal absorbers for application in Alzheimer's disease therapy. We report on hollow gold nanospheres (HAuNS) and gold nanorods (AuNR), which exhibit strong plasmonic near infrared (NIR) absorbance in the optical window of biological tissue and which are functionalized with CLPFFD in two different ways. Therefore the peptide was either directly bound to the particle surface or indirectly to a particle-protecting polyethylene glycol (PEG) ligand shell, thereby reducing the CLPFFD density on the surfaces of both types of particles. Fully PEGylated particles were used for comparison. The effects on cell viability and the fundamental suitability of the HAuNS and AuNR conjugates as photothermal absorbers to inhibit Aß-fibrillation are analysed in vitro. The positive influence of the use of PEG ligands on the reduced cytotoxicity of the conjugates and on the Aß-disaggregation is discussed.

Peptide functionalized magneto-plasmonic nanoparticles obtained by microfluidics for inhibition of ß-amyloid aggregation.

In the present work, we report on the synthesis of peptide functionalized magneto-plasmonic nanoparticles in a simple microfluidic platform. Superparamagnetic nanoparticles and gold nanorods were selected for this study. Magnetic nanoparticles were functionalized with peptide D1, which can bind selectively to toxic aggregates of the ß-amyloid peptide associated with Alzheimer's disease. Gold nanorods were functionalized with chitosan replacing the surfactant cetyltrimethylammonium bromide to reduce the cytotoxic effect. The selected microfluidic strategy yields structures with plasmonic and magnetic properties in a nanostructure. Cytotoxic assays with SH-SY5Y cells demonstrate that nanoparticles obtained by microfluidics do not affect cell viability at the studied concentrations. Additionally, these magneto-plasmonic nanoparticles inhibit fibril formation demonstrating that the magneto-plasmonic nanoparticles obtained by microfluidics could be applied for a potential treatment and diagnosis of Alzheimer's disease.

Photothermal conversion efficiency and cytotoxic effect of gold nanorods stabilized with chitosan, alginate and poly(vinyl alcohol).

Gold nanorods (GNR) use has been proposed in medical applications because of their intrinsic photothermal properties. However, the presence of CTAB molecules adsorbed onto the surface of GNRs results in a highly cytotoxic GNR system. In this work we replace the CTAB molecules with a thiolated chitosan. Once chitosan coated GNRs (Chi-SH-GNR) were attained, a film of alginate (Alg-Chi-SH-GNR) or polyvinyl alcohol (PVA-Chi-SH-GNR) was deposited onto the surface of Chi-GNR by a layer-by-layer process. The photothermal conversion efficiency for the GNR systems was determined irradiating the GNRs suspended in aqua media with a CW 808nm diode laser (CNI, China). The cytotoxicity effect and the photothermal cellular damage of GNR systems were evaluated on a breast cancer cell line. Results show that polymer coats did not affect the transduction photothermal efficiency. Values around 50% were obtained for the different coated gold nanorods. The cytotoxicity of coated gold nanorods diminished significantly compared with those GNR stabilized with CTAB. The laser irradiation of cells treated with gold nanorods showed a decrease in their viability compared with the cells treated but no irradiated.

Metal Nanoparticles as Targeted Carriers Circumventing the Blood-Brain Barrier.

Metal nanoparticles have been proposed as a carrier and a therapeutic agent in biomedical field because of their unique physiochemical properties. Due to these physicochemical properties, they can be used in different fields of biomedicine. In relation to this, plasmonic nanoparticles can be used for detection and photothermal destruction of tumor cells or toxic protein aggregates, and magnetic iron nanoparticles can be used for imaging and for hyperthermia of tumor cells. In addition, both therapy and imaging can be combined in one nanoparticle system, in a process called theranostics. Metal nanoparticles can be synthesized to modulate their size and shape, and conjugated with different ligands, which allow their application in drug delivery, diagnostics, and treatment of central nervous system diseases. This review is focused on the potential applications of metal nanoparticles and their capability to circumvent the blood-brain barrier (BBB). Although many articles have demonstrated delivery of metal nanoparticles to the brain by crossing the BBB after systemic administration, the percentage of the injected dose that reaches this organ is low in comparison to others, especially the liver and spleen. In connection with this drawback, we elaborate the architecture of the BBB and review possible mechanisms to cross this barrier by engineered nanoparticles. The potential uses of metal nanoparticles for treatment of disorders as well as related neurotoxicological considerations are also discussed. Finally, we bring up for discussion a direct and relatively simpler solution to the problem. We discuss this in detail after having proposed the use of the intranasal administration route as a way to circumvent the BBB. This route has not been extensively studied yet for metal nanoparticles, although it could be used as a research tool for mechanistic understanding and toxicity as well as an added value for medical practice.

Interaction of the CLPFFD peptide with gold nanospheres. A Raman, surface enhanced Raman scattering and theoretical study.

In a previous work we demonstrated that toxic aggregates of the protein ß-amyloid (ATAß) involved in the Alzheimer's disease (AD) can be destabilized upon electromagnetic irradiation of the peptide Cys-Leu-Pro-Phe-Phe-Asp (CLPFFD) adsorbed on gold nanospheres (AuNSs). For a selective recognition of the therapeutic target (i.e. ATAß) of AD by the conjugates peptide-nanoparticle it is relevant to understand how the interaction between attached ligands and nanoparticles occurs. In this work a surface enhanced Raman scattering spectroscopy (SERS) study of the interactions of CLPFFD with AuNSs of 10nm average diameter was carried out. The SERS data suggest that phenylalanine displays its aromatic ring coplanar to the surface which is supported by theoretical data obtained from molecular mechanics (MM) and Extended Hückel Theory (EHT) calculations.

Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice.

Gold nanoparticles (GNPs) offer a great promise in biomedicine. Currently, there is no data available regarding the accumulation of nanoparticles in vivo after repeated administration. The purpose of the present study was to evaluate the bioaccumulation and toxic effects of different doses (40, 200, and 400 microg/kg/day) of 12.5 nm GNPs upon intraperitoneal administration in mice every day for 8 days. The gold levels in blood did not increase with the dose administered, whereas in all the organs examined there was a proportional increase on gold, indicating efficient tissue uptake. Although brain was the organ containing the lowest quantity of injected GNPs, our data suggest that GNPs are able to cross the blood-brain barrier and accumulate in the neural tissue. Importantly, no evidence of toxicity was observed in any of the diverse studies performed, including survival, behavior, animal weight, organ morphology, blood biochemistry and tissue histology. The results indicate that tissue accumulation pattern of GNPs depend on the doses administered and the accumulation of the particles does not produce sub-acute physiological damage.

Electrochemical study and analytical applications for new biologically active 2-nitrophenylbenzimidazole derivatives.

The present study addresses the electrochemical behavior and the analytical applications of six 2-nitrophenylbenzimidazole derivatives with activity against Trypanosoma cruzi. When studied in a wide range of pH, by differential pulse polarography, tast polarography and cyclic voltammetry, these compounds exhibited two irreversible cathodic responses. With analytical purposes, the differential pulse polarography mode was selected, which exhibited adequate analytical parameters of repeatability, reproducibility and selectivity. The percentage of recovery was in all cases over 99%, and the detection and quantitation limits were at the level of 1 x 10(-7)mol L(-1) and 1 x 10(-6)mol L(-1), respectively. In addition, the differential pulse polarography method was successfully applied to study the hydrolytic degradation kinetic of one of the tested compounds. Activation energy, kinetic rate constants at different temperatures and half-life values of such application are reported.

Fatty acyl moieties: improving Pro-rich peptide uptake inside HeLa cells.

In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro-rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro-rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.

Self-assembly of the amphipathic helix (VHLPPP)8. A mechanism for zein protein body formation.

gamma-Zein, a maize storage protein with an N-terminal proline-rich repetitive domain (gamma-ZNPRD), is located at the periphery of protein bodies. This domain appears to be indispensable for the aggregation of the protein on the surface of the organelle. The peptide (VHLPPP)8, spanning the gamma-ZNPRD, adopts a polyproline II (PPII) conformation that gives an amphipathic helix different from the alpha-helix. We used atomic force microscopy to study the surface organisation of the octamer, and transmission electron microscopy to visualise aggregates of the peptide in aqueous solution. We consider two self-assembly patterns that take account of the observed features. The micellar one fits best with the experimental results presented. Moreover, we found that this peptide has properties associated with surfactants, and form micelles in solution. This spontaneous amphipathic arrangement of the gamma-ZNPRD suggests a mechanism of gamma-zein deposition inside maize protein bodies.

Occurrence of cadaverine in hairy roots of Brugmansia candida.

The polyamine, cadaverine, was detected in transformed root cultures of Brugmansia candida (syn. Datura candida), a Solanaceae which produces the tropane alkaloids scopolamine and hyoscyamine. To the best of our knowledge, this is the first time that the existence of this uncommon polyamine has been detected in a Datura species. Cadaverine, however, could not be found in the whole plant. The occurrence of cadaverine in hairy roots could be a consequence of either the transformation or a response to stress. Also, cadaverine could be participating in other secondary pathways rather than to the tropane alkaloids. The common polyamines, putrescine, spermidine and spermine were also observed.

Exploring the conformation of bilirubins with natural and unnatural analogues: use of positional and bridged isomers of bilirubin IXalpha.

Unlike bilirubin IXalpha (1), the isomers bilirubin IXdelta (2) and neobilirubin IXbeta (3) do not require conjugation with glucuronic acid in order to be excreted. A conformational analysis employing an optimized Monte Carlo method and a mixed Monte Carlo stochastic dynamics reveals that isomer 2 exhibits a structure more closed than the well known 'ridge-tile' conformation of 1. The change in the position of both propionic acid chains causes the loss of at least four hydrogen bonds. On the other hand, the change in the configuration of the distal dipyrrinone and the blockage of the lactamic nitrogen by the presence of a bridge in isomer 3 results in an open and more elongated structure, where the chance of hydrogen bond formation in this region is obliterated. The resulting molecular models for these compounds are consistent with 1H NM R, UV-vis, and TLC data.

Probing the conformation of bilirubins with monopropionic analogs: a biological, spectroscopic, and molecular modeling study.

The in vivo metabolism of a bilirubin analog substituted with a propionic acid chain in C8 (5) showed that it is excreted in bile conjugated with glucuronic acid, while a positional isomer substituted with a propionate in C7 (6) is excreted in bile without conjugation. A conformational analysis employing an optimized Monte Carlo method and a mixed Monte Carlo/stochastic dynamics reveals that isomer 5 adopts a 'ridge tile' conformation, stabilized by the presence of three intramolecular hydrogen bonds. On the contrary, isomer 6 exhibits a more closed structure, where impairment in the formation of at least one of the hydrogen bonds occurs. These theoretical predictions agree well with 1H NMR, UV-vis, and TLC data.

Confirmation of Syva enzyme multiple immunoassay technique (EMIT) d.a.u. and Roche Abuscreen radioimmunoassay (RIA) (125I) urine cannabinoid immunoassays by gas chromatographic/mass spectrometric (GC/MS) and bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) methods.

Thirty human urines screened positive by the Syva enzyme multiple immunoassay technique (EMIT) d.a.u. urine cannabinoid assay were also positive for the major marijuana urinary metabolite 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) when assayed by gas chromatographic/mass spectrometric (GC/MS) and a noninstrumental qualitative bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) technique. The noninstrumental BPA-TLC procedure was the simpler of the two techniques to perform and interpret. Assay of these same samples by the Roche Abuscreen radioimmunoassay (RIA) for cannabinoids (125I) revealed that reliance on the 100-ng/mL equivalent positive calibrator yielded a high incidence of false negative results (10 out of 30). The performance of these same 4 assays on 30 true negatives also was evaluated. All samples were negative for cannabinoids by EMIT and RIA, and for THC-COOH by BPA-TLC. GC/MS assay, however, detected spurious low levels of approximately 5-ng/mL THC-COOH in two instances. Because of this, a reliability level of 10 ng/mL was set for the routine quantitative confirmation of THC-COOH by the GC/MS method.

Confirmation of EMIT cannabinoid assay results by bonded phase adsorption with thin layer chromatography.

Clinical urine specimens were screened for the presence of cannabinoids using the EMIT Cannabinoid Assay. Aliquots of these samples were also analyzed for 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THCA), the major cannabis metabolite in urine, by a technique which combines bonded phase adsorption (BPA) and thin layer chromatography (TLC). A 100% agreement between EMIT and BPA-TLC results was observed when at least 20 mL of urine was assayed by BPA-TLC. Bonded phase adsorption coupled with thin layer chromatography appears to be a suitable technique for the confirmation of positive EMIT Cannabinoid Assay results.

Quantitative determination of haloperidol in human plasma by high-performance liquid chromatography.

We present a method for the quantitative determination of haloperidol in human plasma. The high-performance liquid chromatographic method of analysis is a significant advancement in terms of ease and speed of haloperidol determination. The drug and the internal standard, chlorohaloperidol, are extracted from 2.0 ml of serum or plasma, back-extracted into the aqueous mobile phase, separated on a C-18 reversed-phase column, and monitored at 254 nm. The potential interference by other drugs was evaluated and was found to be negative. The method is sensitive to at least 5 ng/ml of extracted material and is suitable for drug measurement in the therapeutic range (5-20 ng/ml) and in the toxic range (greater than 50 ng/ml).

Estimation of phencyclidine in leafy mixtures and in vapor phase after smoking.

Five street samples of leafy material coated with phencyclidine (PCP) were analyzed by a gas chromatographic nitrogen detection assay. The samples contained 15.6+1.8% PCP by weight or 32.2+ 13.8 mg PCP per "joint". An aliquot of a joint was smoked with a laboratory apparatus and the vaporized PCP was collected on a filter. Only 22.6+8.0% of the PCP or 6.7+2.1 mg PCP, reached the filter. This amount is in an approximation of the dose of PCP which becomes available to the oral and pulmonary mucosa following the smoking of a single PCP coated joint.

Phencyclidine-induced stereotype in rats: effects of methadone, apomorphine, and naloxone.

Phencyclidine (PCP) given to male Wistar rats produced hyperactivity and various stereotypic motor behaviors. Methadone, apomorphine, and naloxone were tested for their effects on PCP-induced stereotypy. Methadone (0.5 mg/kg) had no effect on the hyperactivity produced by PCP, but significantly attenuated PCP-induced stereotypy when given both before and after PCP. Low doses of apomorphine were equally effective as methadone in attenuating PCP-induced stereotypy. However, when naloxone was given after methadone or apomorphine to PCP-treated rats, the full PCP-induced stereotypy was again observed. Naloxone pretreatment on doses up to 20 mg/kg was not effective in antagonizing PCP-induced behavioral effects. Methadone and apomorphine antagonism of PCP-induced stereotypy may be mediated by opiate receptors. The results of this study and observations from human studies collectively suggest the possible effectiveness of opiates in treating PCP-induced and functional psychoses.