Gold Nanoprobes for Detection of a Crucial EGFR Deletion for Early Diagnosis of Non-Small-Cell Lung Cancer.

Gold nanoparticles (AuNPs) exhibit improved optical and spectral properties compared to bulk materials, making them suitable for the detection of DNA, RNA, antigens, and antibodies. Here, we describe a simple, selective, and rapid non-cross linking detection assay, using approx. 35 nm spherical Au nanoprobes, for a common mutation occurring in exon 19 of the epidermal growth factor receptor (EGFR), associated with non-small-cell lung cancer cells. AuNPs were synthesized based on the seed-mediated growth method and functionalized with a specific 16 bp thiolated oligonucleotide using a pH-assisted method. Both AuNPs and Au nanoprobes proved to be highly stable and monodisperse through ultraviolet-visible spectrophotometry, dynamic light scattering (DLS), and electrophoretic light scattering (ELS). Our results indicate a detection limit of 1.5 µg mL-1 using a 0.15 nmol dm-3 Au nanoprobe concentration. In conclusion, this work presents an effective possibility for a straightforward, fast, and inexpensive alternative for the detection of DNA sequences related to lung cancer, leading to a potential platform for early diagnosis of lung cancer patients.

Fe3O4-Au Core-Shell Nanoparticles as a Multimodal Platform for In Vivo Imaging and Focused Photothermal Therapy.

In this study, we report the synthesis of gold-coated iron oxide nanoparticles capped with polyvinylpyrrolidone (Fe@Au NPs). The as-synthesized nanoparticles (NPs) exhibited good stability in aqueous media and excellent features as contrast agents (CA) for both magnetic resonance imaging (MRI) and X-ray computed tomography (CT). Additionally, due to the presence of the local surface plasmon resonances of gold, the NPs showed exploitable "light-to-heat" conversion ability in the near-infrared (NIR) region, a key attribute for effective photothermal therapies (PTT). In vitro experiments revealed biocompatibility as well as excellent efficiency in killing glioblastoma cells via PTT. The in vivo nontoxicity of the NPs was demonstrated using zebrafish embryos as an intermediate step between cells and rodent models. To warrant that an effective therapeutic dose was achieved inside the tumor, both intratumoral and intravenous routes were screened in rodent models by MRI and CT. The pharmacokinetics and biodistribution confirmed the multimodal imaging CA capabilities of the Fe@AuNPs and revealed constraints of the intravenous route for tumor targeting, dictating intratumoral administration for therapeutic applications. Finally, Fe@Au NPs were successfully used for an in vivo proof of concept of imaging-guided focused PTT against glioblastoma multiforme in a mouse model.

Study of the intestinal uptake and permeability of gold nanoparticles using both in vitro and in vivo approaches.

Gold nanoparticles (AuNPs) are highly attractive to biomedical applications. Here, we investigated the effects of (i) ca. 15 nm spherical AuNPs capped with citrate or 11-mercaptoundecanoic acid (MUA) and (ii) ca. 60 nm spherical citrate-capped AuNPs, and ca. 60 nm MUA-capped star-shaped AuNPs on the cytotoxicity, cellular uptake and permeability, using media supplemented or not with 1% fetal bovine serum (FBS) on caucasian colon adenocarcinoma Caco-2 cells. In addition, the colloidal stability of the nanoparticles in media (supplemented or not) was assessed after 24 h-incubations at 60 µM. The 60 nm gold nanospheres and stars were administrated orally to Wistar rats in order to evaluate their systemic absorption and biodistribution after 24 h. At non-supplemented media settings, citrate-capped gold nanoparticles seem to be more toxic than their MUA-capped counterparts. Also, smaller nanoparticles show higher toxicity than larger ones. The use of cell culture media with 1% FBS not only increased the stability of all AuNPs, as also significantly reduced their cytotoxicity. In the uptake studies, higher AuNPs incorporation was noticed in serum supplemented media, this effect being particularly significant for the 60 nm nanoparticles. Cellular incorporation depended also on the capping agent and size. None of the tested samples crossed the in vitro intestinal barrier. Confirming the in vitro results, the in vivo biodistribution study of the 60 nm AuNPs orally given to rats showed that their systemic absorption is low and that they are mainly eliminated through the faeces. Altogether, these preliminary results suggest that our novel AuNPs have high potential to be considered promising candidates for application in diagnostics or drug delivery at the intestinal level, showing high biocompatibility. However, unless it is desired that these nanomaterials avoid systemic absorption upon oral administration, additional functionalization should be sought to increase their low bioavailability.

Synthesis and Characterization of Elongated-Shaped Silver Nanoparticles as a Biocompatible Anisotropic SERS Probe for Intracellular Imaging: Theoretical Modeling and Experimental Verification.

Progress in the field of biocompatible SERS nanoparticles has promising prospects for biomedical applications. In this work, we have developed a biocompatible Raman probe by combining anisotropic silver nanoparticles with the dye rhodamine 6G followed by subsequent coating with bovine serum albumin. This nanosystem presents strong SERS capabilities in the near infrared (NIR) with a very high (2.7 × 107) analytical enhancement factor. Theoretical calculations reveal the effects of the electromagnetic and chemical mechanisms in the observed SERS effect for this nanosystem. Finite element method (FEM) calculations showed a considerable near field enhancement in NIR. Using density functional quantum chemical calculations, the chemical enhancement mechanism of rhodamine 6G by interaction with the nanoparticles was probed, allowing us to calculate spectra that closely reproduce the experimental results. The nanosystem was tested in cell culture experiments, showing cell internalization and also proving to be completely biocompatible, as no cell death was observed. Using a NIR laser, SERS signals could be detected even from inside cells, proving the applicability of this nanosystem as a biocompatible SERS probe.

Improvement in left intraventricular pressure gradients after aortic valve replacement in aortic stenosis patients.

NEW FINDINGS: What is the central question of this study? Normal diastolic and systolic intraventricular pressure gradients are decreased when left ventricular filling and/or emptying are compromised. We hypothesized that in patients with severe aortic valve stenosis, a condition that interferes with ventricular filling and emptying, those gradients would be disturbed. What is the main finding and its importance? We showed the existence of intraventricular pressure gradients throughout the cardiac cycle in the human left ventricle. Moreover, we demonstrated, for the first time, that diastolic and systolic gradients, which are markers of normal ventricular filling and emptying, respectively, improved in patients with severe aortic valve stenosis immediately after valve replacement. The present study was conducted to characterize left intraventicular pressure gradients, which are markers of normal cardiac function, in patients with severe aortic stenosis, a condition that interferes with ventricular filling and emptying. In 10 patients (four male; mean age 71.3 ± 4.8 years old) undergoing aortic valve replacement, two high-fidelity pressure catheters were inserted inside the cavity of the left ventricle through an apical puncture and positioned in the apex and outflow tract below the aortic valve. Pressures were continuously acquired and gradients calculated as apical minus outflow tract pressure, before and immediately after aortic valve replacement. During early filling, we recorded a negative intraventricular gradient along the basal portion of the left ventricle in the apical direction (-0.82 ± 0.45 mmHg), which increased to -3.97 ± 0.42 mmHg after aortic valve replacement. In late filling, intraventricular flow was now directed towards the outflow tract, with a positive pressure gradient both before (+1.23 ± 0.37 mmHg) and after surgery (+2.12 ± 0.58 mmHg). During systole, before surgery we observed a positive pressure gradient between the apex and outflow tract during both rapid (+1.60 ± 0.21 mmHg) and slow ejection phases (+1.68 ± 0.12 mmHg), whereas after aortic valve replacement the positive gradient (+1.54 ± 0.15 mmHg) during rapid ejection was inverted (-3.92 ± 0.34 mmHg) during the slow ejection phase. We demonstrated that in patients with severe aortic stenosis both diastolic and systolic intraventricular pressure gradients are significantly attenuated but can be restored immediately after aortic valve replacement. The assessment and measurement of intraventricular pressure gradients and their modulation in pathophysiological conditions may provide novel insights into cardiac physiology.

In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests.

Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5-300 µg ml(-1)), prepared in complete and serum-free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical-chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell-free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid-coated ION were less cytotoxic than silica-coated ION; besides, a serum-protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications.

What has changed in surgical treatment of severe aortic stenosis with the advent of percutaneous intervention?

INTRODUCTION AND OBJECTIVE: Transcatheter aortic valve implantation (TAVI) is an alternative therapeutic approach to patients not considered suitable for surgical aortic valve replacement (SAVR) due to their high operative risk. We sought to assess the impact of TAVI on the profile and operative results of patients with severe aortic stenosis undergoing SAVR. METHODS: A total of 214 patients were included, of whom 103 consecutive patients underwent isolated SAVR in 2005 and 111 in 2009. Patients' demographic and operative data were collected retrospectively. Operative and one-year mortality and morbidity were analyzed. RESULTS: Patients' mean age was 70 years, and 56% were female. Following the introduction of a TAVI program, patients undergoing conventional surgery were older, with more comorbidities. Overall 30-day and one-year mortality were 2.8% and 7.0%, respectively. After the introduction of TAVI, the observed mortality rate for SAVR decreased, but not significantly (operative mortality: 3.9% before TAVI vs. 1.8% after TAVI, p = NS; one-year mortality: 10% vs. 4.5%, p = NS). Striking differences were observed in morbidity (operative morbidity: 23.3% before TAVI vs. 13.5% after TAVI, p = 0.047, and one-year morbidity: 20.4% vs. 9.9%, p = 0.032). CONCLUSIONS: Since the introduction of a TAVI program at our center, the number of patients undergoing SAVR has increased, with a slight rise in surgical risk, but without worsening the final operative results. The implementation of a TAVI program has thus had a positive impact on the volume of procedures, patient selection and outcomes in SAVR.

Incremental value of adenosine stress cardiac magnetic resonance in coronary artery disease detection.

INTRODUCTION: Cardiac magnetic resonance myocardial perfusion imaging (CMR-MPI) is considered a state-of-the-art non-invasive modality for ischemia detection but its additive value in a multiple-test strategy in patients with suspected coronary artery disease (CAD) is not fully validated. We aimed to evaluate CMR-MPI integration with exercise treadmill test (ETT) for the diagnostic workup of patients with suspected CAD, having invasive fractional flow reserve (FFR) as reference standard. METHODS: In this prospective single-center study, patients with suspected CAD underwent sequential ETT, CMR-MPI and X-ray invasive coronary angiography (XA). Significant CAD was defined by the presence of stenosis >40% with FFR ≤ 0.8 in vessels >2 mm or ≥90% stenosis/occlusion. RESULTS: 80 symptomatic patients (68% male, 61 ± 8 years) were enrolled. Compared to ETT, CMR-MPI showed similar sensitivity (81%) and higher specificity (93 vs. 58%, p < 0.001) for CAD detection (prevalence = 46%) translating into better diagnostic performance (AUC 0.87 vs. 0.70; p = 0.002). CMR-MPI improved accuracy independently of ETT in all patients with high pre-test probability and in intermediate-probability patients but those with a clearly positive-ETT (symptoms + ST-shift), in whom ETT correctly identified CAD. In the low-probability group CMR-MPI was useful as a gatekeeper for XA after a positive-ETT. The best integrating protocol achieved a global accuracy of 89% (AUC 0.88) and was clearly superior to an approach based solely in ETT (AUC 0.70, p < 0.001), yet similar to isolated CMR-MPI (AUC 0.87, p = ns). CONCLUSIONS: CMR-MPI has high sensitivity and specificity for CAD detection and may be combined with ETT in a diagnostic workflow aiming to increase accuracy and reduce the number of unnecessary catheterizations.

Influence of the surface coating on the cytotoxicity, genotoxicity and uptake of gold nanoparticles in human HepG2 cells.

The toxicological profile of gold nanoparticles (AuNPs) remains controversial. Significant efforts to develop surface coatings to improve biocompatibility have been carried out. In vivo biodistribution studies have shown that the liver is a target for AuNPs accumulation. Therefore, we investigated the effects induced by ~20 nm spherical AuNPs (0-200 µM Au) with two surface coatings, citrate (Cit) compared with 11-mercaptoundecanoic acid (11-MUA), in human liver HepG2 cells. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays after 24 to 72 h of incubation. DNA damage was assessed by the comet assay, 24 h after incubation with the capped AuNPs. Uptake and subcellular distribution of the tested AuNPs was evaluated by quantifying the gold intracellular content by graphite furnace atomic absorption spectrometry (GFAAS) and transmission electron microscopy (TEM), respectively. The obtained results indicate that both differently coated AuNPs did not induce significant cytotoxicity. An inverse concentration-dependent increase in comet tail intensity and tail moment was observed in Cit-AuNPs- but not in MUA-AuNPs-exposed cells. Both AuNPs were internalized in a concentration-dependent manner. However, no differences were found in the extent of the internalization between the two types of NPs. Electron-dense deposits of agglomerates of Cit- and MUA-AuNPs were observed either inside endosomes or in the intercellular spaces. In spite of the absence of cytotoxicity, DNA damage was observed after exposure to the lower concentrations of Cit- but not to MUA-AuNPs. Thus, our data supports the importance of the surface properties to increase the biocompatibility and safety of AuNPs.

Aborted sudden cardiac death as first presentation of Wolff-Parkinson-White syndrome.

Sudden cardiac death (SCD) can be the first clinical manifestation of Wolff-Parkinson-White (WPW) syndrome. Catheter ablation of accessory pathways is now a safe and effective procedure, and is widely recommended in patients with WPW syndrome. However, management of the asymptomatic WPW patient remains controversial. Recent studies have readdressed the issue of risk stratification and prophylactic catheter ablation. We describe a case of malignant arrhythmia and aborted SCD as first presentation of WPW syndrome in a previously asymptomatic 17-year-old patient.

Solution and biological behaviour of enrofloxacin metalloantibiotics: a route to counteract bacterial resistance?

Solution behaviour of enrofloxacin complexes with copper(II), nickel(II), cobalt(II) and zinc(II) in the presence and absence of 1, 10-phenanthroline was studied in aqueous solution, by potentiometry. The results obtained show that under physiological conditions (micromolar concentration range and pH 7.4) only copper(II) forms stable complexes. Binary copper(II)/enrofloxacin and ternary copper(II)/enrofloxacin/phenanthroline complexes were synthesised and characterized by elemental analysis, UV-visible spectroscopy and FTIR. The antimicrobial activity of these complexes and of copper(II)/enrofloxacin and copper(II)/enrofloxacin/phenanthroline solutions, prepared by mixing of the individual components in the same stoichiometric proportion and concentration range used for the synthesised complexes, was tested against two different Escherichia coli strains. Although, at a glance, the results point to a possible use of both complexes as metalloantibiotics, a detailed analysis shows that, at biological concentrations, the copper(II) binary complex does not exist and the antimicrobial activity observed is a consequence of its dissociation into free enrofloxacin. Consequently, only the ternary complex seems worth pursuing as a possible antimicrobial agent candidate. Moreover, as the biological studies showed, both the synthesised complexes and the solutions prepared by mixing the components exhibited the same behaviour. Hence, a new, faster and accurate methodology to screen metalloantibiotics prior to synthesis of the complexes is proposed.

AFM and electron microscopy study of the unusual aggregation behavior of metallosurfactants based on iron(II) complexes with bipyridine ligands.

The aggregation behavior in water-rich solutions of five iron(II) complexes with alkylated derivatives of 2,2'-bipyridine was studied by electron microscopy (cryo-SEM, SEM, and TEM) and AFM. The results obtained by cryo-SEM on frozen colloidal solutions show that the morphology of the aggregates strongly depends on the length of the alkyl chains in the bipyridine ligands, with shorter alkyl chains forming rod-like structures, whereas for compounds with longer alkyl chains, only spherical structures were detected. The self-aggregates were further characterized by SEM and TEM. The results show that their overall morphology depends only on the length of the alkyl chain of the bipyridine ligands and that the samples show a broad size distribution. In addition, TEM and SEM were used to study the stability of the self-aggregates in solution, the effect of addition of methanol, and the temperature used in the preparation of the colloidal solutions. AFM studies of the aggregates either dried in ambient conditions or dehydrated by long drying under vacuum showed partially collapsed self-aggregates in the latter case, showing that the aggregates contain water in their core, indicating that the self-aggregation leads to vesicle-type structures.

Cytotoxic activity of metal complexes of biogenic polyamines: polynuclear platinum(II) chelates.

Several polynuclear Pt(II) chelates with biogenic polyamines were synthesized and screened for their potential antiproliferative and cytotoxic activity in different human cancer cell lines. To gather information regarding the structure-activity relationships underlying their biological activity, the complexes studied were designed to differ in geometrical parameters such as the nature of the ligand and the number and chemical environment of the metal centers. Distinct effects were found for different cell lines and different structural characteristics of the complexes; chelates II, III, and IV displayed specificity toward the HeLa and HSC-3 epithelial-type cells, while V, VI, and VII were clearly more effective against the THP-1, MOLT-3, and CCRF-CEM leukemia cell lines. The toxicity of these Pt(II) complexes on noncancer cells was, in all cases, found to be reversed upon drug removal.