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.

Cellular uptake and toxicity of gold nanoparticles on two distinct hepatic cell models.

Gold nanoparticles (AuNPs) have huge potential for various biomedical applications, but their successful use depends on their uptake and possible toxicity in the liver, their main site for accumulation. Therefore, in this work we compared the cytotoxic effects induced by AuNPs with different size (~ 15 nm and 60 nm), shape (nanospheres and nanostars) and capping [citrate- or 11-mercaptoundecanoic acid (MUA)], in human HepaRG cells or primary rat hepatocytes (PRH) cultivated with serum-free or Foetal Bovine Serum (FBS)-supplemented media. The safety assessment of the AuNPs demonstrated that overall they present low toxicity towards hepatic cells. Among all the tested AuNPs, the smaller 15 nm spheres displayed the highest toxicity. The toxicological effect was capping, size and cell-type dependent with citrate-capping more toxic than MUA (PRH with FBS), the 15 nm AuNPs more toxic than 60 nm counterparts and PRH more sensitive, as compared to the HepaRG cells. The incubation with FBS-free media produced aggregation of AuNPs while its presence greatly influenced the toxicity outcomes. The cellular uptake of AuNPs was shape, size and capping dependent in PRH cultivated in FBS-supplemented media, and significantly different between the two types of cells with extensively higher internalization of AuNPs in PRH, as compared to the HepaRG cells. These data show that the physical-chemical properties of AuNPs, including size and shape, as well as the type of cellular model, greatly influence the interaction of the AuNPs with the biological environment and consequently, their toxicological effects.

Gold Nanoparticles Induce Oxidative Stress and Apoptosis in Human Kidney Cells.

Gold nanoparticles (AuNPs) are highly attractive for biomedical applications. Therefore, several in vitro and in vivo studies have addressed their safety evaluation. Nevertheless, there is a lack of knowledge regarding their potential detrimental effect on human kidney. To evaluate this effect, AuNPs with different sizes (13 nm and 60 nm), shapes (spheres and stars), and coated with 11-mercaptoundecanoic acid (MUA) or with sodium citrate, were synthesized, characterized, and their toxicological effects evaluated 24 h after incubation with a proximal tubular cell line derived from normal human kidney (HK-2). After exposure, viability was assessed by the MTT assay. Changes in lysosomal integrity, mitochondrial membrane potential (ΔΨm), reactive species (ROS/RNS), intracellular glutathione (total GSH), and ATP were also evaluated. Apoptosis was investigated through the evaluation of the activity of caspases 3, 8 and 9. Overall, the tested AuNPs targeted mainly the mitochondria in a concentration-dependent manner. The lysosomal integrity was also affected but to a lower extent. The smaller 13 nm nanospheres (both citrate- and MUA-coated) proved to be the most toxic among all types of AuNPs, increasing ROS production and decreasing mitochondrial membrane potential (p ≤ 0.01). For the MUA-coated 13 nm nanospheres, these effects were associated also to increased levels of total glutathione (p ≤ 0.01) and enhanced ATP production (p ≤ 0.05). Programmed cell death was detected through the activation of both extrinsic and intrinsic pathways (caspase 8 and 9) (p ≤ 0.05). We found that the larger 60 nm AuNPs, both nanospheres and nanostars, are apparently less toxic than their smaller counter parts. Considering the results herein presented, it should be taken into consideration that even if renal clearance of the AuNPs is desirable, since it would prevent accumulation and detrimental effects in other organs, a possible intracellular accumulation of AuNPs in kidneys can induce cell damage and later compromise kidney function.

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.

3,4-Methylenedioxymethamphetamine Hepatotoxicity under the Heat Stress Condition: Novel Insights from in Vitro Metabolomic Studies.

Hyperthermia has been extensively reported as a life-threatening consequence of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse. In this work, we used a sensitive untargeted metabolomic approach based on gas chromatography-mass spectrometry to evaluate the impact of hyperthermia on the hepatic metabolic changes caused by MDMA. For this purpose, primary mouse hepatocytes were exposed to subtoxic (LC01 and LC10) and toxic (LC30) concentrations of MDMA for 24 h, at 37 or 40.5 °C (simulating body temperature increase after MDMA consumption), and alterations on both intracellular metabolome and extracellular volatilome were evaluated. Multivariate analysis showed that metabolic patterns clearly discriminate MDMA treated cells from control cells, both in normothermic and hyperthermic conditions. The metabolic signature was found to be largely common to MDMA subtoxic and toxic concentrations, although with evident differences in the magnitude of response, with metabolic changes significantly more pronounced at 40.5 °C. Discriminant metabolites associated with MDMA-induced hepatotoxicity are mostly involved in the amino acid metabolism, aminoacyl tRNA biosynthesis, glutathione metabolism, tricarboxylic acid cycle, and pyruvate metabolism. Moreover, our metabolomic findings were corroborated by classical toxicity parameters, demonstrating the high sensitivity of this omic approach to assess molecular-level effects. Overall, this study indicates that MDMA triggers significant metabolic alterations on hepatic cells, even at low concentrations, that are clearly exacerbated at high temperatures. These findings provide new metabolic pieces to solve the puzzle of MDMA's hepatotoxicity mechanism and emphasize the increased risks of MDMA abuse due to the thermogenic action of the drug.

A Metabolomic Approach for the In Vivo Study of Gold Nanospheres and Nanostars after a Single-Dose Intravenous Administration to Wistar Rats.

Gold nanoparticles (AuNPs) are promising nanoplatforms for drug therapy, diagnostic and imaging. However, biological comparison studies for different types of AuNPs fail in consistency due to the lack of sensitive methods to detect subtle differences in the expression of toxicity. Therefore, innovative and sensitive approaches such as metabolomics are much needed to discriminate toxicity, specially at low doses. The current work aims to compare the in vivo toxicological effects of gold nanospheres versus gold nanostars (of similar ~40 nm diameter and coated with 11-mercaptoundecanoic acid) 24 h after an intravenous administration of a single dose (1.33 × 1011 AuNPs/kg) to Wistar rats. The biodistribution of both types of AuNPs was determined by graphite furnace atomic absorption spectroscopy. The metabolic effects of the AuNPs on their main target organ, the liver, were analyzed using a GC-MS-based metabolomic approach. Conventional toxicological endpoints, including the levels of ATP and reduced and oxidized glutathione, were also investigated. The results show that AuNPs preferentially accumulate in the liver and, to a lesser extent, in the spleen and lungs. In other organs (kidney, heart, brain), Au content was below the limit of quantification. Reduced glutathione levels increased for both nanospheres and nanostars in the liver, but ATP levels were unaltered. Multivariate analysis showed a good discrimination between the two types of AuNPs (sphere- versus star-shaped nanoparticles) and compared to control group. The metabolic pathways involved in the discrimination were associated with the metabolism of fatty acids, pyrimidine and purine, arachidonic acid, biotin, glycine and synthesis of amino acids. In conclusion, the biodistribution, toxicological, and metabolic profiles of gold nanospheres and gold nanostars were described. Metabolomics proved to be a very useful tool for the comparative study of different types of AuNPs and raised awareness about the pathways associated to their distinct biological effects.

Hepatic Metabolic Derangements Triggered by Hyperthermia: An In Vitro Metabolomic Study.

BACKGROUND AND AIMS: Liver toxicity is a well-documented and potentially fatal adverse complication of hyperthermia. However, the impact of hyperthermia on the hepatic metabolome has hitherto not been investigated. METHODS: In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to assess the in vitro metabolic response of primary mouse hepatocytes (PMH, n = 10) to a heat stress stimulus, i.e., after 24 h exposure to 40.5 °C. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed. RESULTS: Multivariate analysis showed alterations in levels of 22 intra- and 59 extracellular metabolites, unveiling the capability of the metabolic pattern to discriminate cells exposed to heat stress from cells incubated at normothermic conditions (37 °C). Hyperthermia caused a considerable loss of cell viability that was accompanied by significant alterations in the tricarboxylic acid cycle, amino acids metabolism, urea cycle, glutamate metabolism, pentose phosphate pathway, and in the volatile signature associated with the lipid peroxidation process. CONCLUSION: These results provide novel insights into the mechanisms underlying hyperthermia-induced hepatocellular damage.

A multiparametric study of gold nanoparticles cytotoxicity, internalization and permeability using an in vitro model of blood-brain barrier. Influence of size, shape and capping agent.

Gold nanoparticles (AuNPs) have biomedical application on imaging and due to increased optical performance, star-shaped AuNPs are of special interest. Because shape, size and capping greatly influence their toxicokinetics and toxicodynamics, a systematic multiparametric comparative study of the influence of these parameters on the cytotoxicity, internalization, and in vitro permeability was conducted in human Cerebral Microvascular Endothelial Cell line (hCMEC/D3), an in vitro model of the human blood-brain barrier (BBB). AuNPs of different size (14 nm and ∼50 nm), shape (spheres and stars), and coating (11-mercaptoundecanoic acid or MUA and sodium citrate) were synthesized and fully characterized. The time- and concentration-dependent cytotoxic profile of the tested AuNPs differed for the different AuNPs. Generally, toxicity was greater for stars relative to sphere-shaped AuNPs, and citrate coating was more toxic than MUA. Regarding the influence of size, smaller-sized AuNPs were more cytotoxic when compared at the same Au concentration. However, when the concentration of AuNPs was expressed as the number of AuNPs/mL, a higher degree of cytotoxicity was noted for the larger ̴50 nm AuNPs. To understand the influence of size, shape and capping, a systematic study design, in which only one of the variables changes, is determinant for correct data interpretation. Considering the results herein presented, for the sake of comparison of differently-sized AuNPs, it is preferable to design the study based upon the number of nanoparticles, since at a fixed Au concentration the number of particles available to promote effect is higher for smaller-sized AuNPs. Cellular internalization also differed among the tested AuNPs; although all were unable to cross the in vitro BBB, the intracellularly accumulated AuNPs can induce cell damage and later compromise BBB integrity and permeability.

Toxicological Evaluation of Luminescent Silica Nanoparticles as New Drug Nanocarriers in Different Cancer Cell Lines.

Luminescent mesoporous silica nanoparticles, CdTeQDs@MNs@PEG1, SiQDs@Isoc@MNs and SiQDs@Isoc@MNs@PEG2, were successfully synthetized and characterized by SEM, TEM, XRD, N2 nitrogen isotherms, ¹H NMR, IR, absorption, and emission spectroscopy. Cytotoxic evaluation of these nanoparticles was performed in relevant in vitro cell models, such as human hepatoma HepG2, human brain endothelial (hCMEC/D3), and human epithelial colorectal adenocarcinoma (Caco-2) cell lines. None of the tested nanoparticles showed significant cytotoxicity in any of the three performed assays (MTT/NR/ LDH) compared with the respective solvent and/or coating controls, excepting for CdTeQDs@MNs@PEG1 nanoparticles, where significant toxicity was noticed in hCMEC/D3 cells. The results presented reveal that SiQDs-based mesoporous silica nanoparticles are promising nanoplatforms for cancer treatment, with a pH-responsive drug release profile and the ability to load 80% of doxorubicin.

Ethanol addictively enhances the in vitro cardiotoxicity of cocaine through oxidative damage, energetic deregulation, and apoptosis.

Cocaine (COC) is frequently consumed in polydrug abuse settings, and ethanol (EtOH) is the most prominent co-abused substance. Clinical data and experimental evidence suggest that the co-administration of COC with EtOH can be more cardiotoxic than EtOH or COC alone, but information on the molecular pathways involved is scarce. Since these data are crucial to potentiate the identification of therapeutic targets to treat intoxications, we sought to (i) elucidate the type of interaction that occurs between both substances, and (ii) assess the mechanisms implicated in the cardiotoxic effects elicited by COC combined with EtOH. For this purpose, H9c2 cardiomyocytes were exposed to COC (104 µM-6.5 mM) and EtOH (977 µM-4 M), individually or combined at a molar ratio based on blood concentrations of intoxicated abusers (COC 1: EtOH 9; 206 µM-110 mM). After 24 h, cell metabolic viability was recorded by the MTT assay and mixture toxicity expectations were calculated using the independent action (IA) and concentration addition (CA) models. EtOH (EC50 305.26 mM) proved to act additively with COC (EC50 2.60 mM) to significantly increase the drug in vitro cardiotoxicity, even when both substances were combined at individually non-cytotoxic concentrations. Experimental mixture testing (EC50 19.18 ± 3.36 mM) demonstrated that the cardiotoxicity was fairly similar to that predicted by IA (EC50 22.95 mM) and CA (EC50 21.75 mM), supporting additivity. Concentration-dependent increases of intracellular ROS/RNS and GSSG, depletion of GSH and ATP, along with mitochondrial hyperpolarization and activation of intrinsic, extrinsic, and common apoptosis pathways were observed both for single and combined exposures. In general, the mixture exhibited a toxicological profile that mechanistically did not deviate from the single drugs, suggesting that interventions such as antioxidant administration might aid in the clinical treatment of this type of polydrug intoxication. In a clinical perspective, the observed additive mixture effect may reflect the increased hazards at which users of this combination are exposed to in recreational settings.

[Estimation of uncertainty of measurement in clinical biochemistry]. / Incertitudinea de masurare. Posibilitati de estimare in masuratorile cantitative din laboratoarele de analize medicale.

The uncertainty of measurement (UM) or measurement uncertainty is known as the parameter associated with the result of a measurement. Repeated measurements usually reveal slightly different results for the same analyte, sometimes a little higher, sometimes a little lower, because the results of a measurement are depending not only by the analyte itself, but also, by a number of error factors that could give doubts about the estimate. The uncertainty of the measurement represent the quantitative, mathematically expression of this doubt. UM is a range of measured values which is probably to enclose the true value of the measured. Calculation of UM for all types of laboratories is regularized by the ISO Guide to the Expression of Uncertainty in Measurement (abbreviated GUM) and the SR ENV 13005 : 2003 (both recognized by European Accreditation). Even if the GUM rules about UM estimation are very strictly, the offering of the result together with UM will increase the confidence of customers (patients or physicians). In this study the authors are presenting the possibilities of UM assessing in labs from our country by using the data obtained in the procedures of methods validation, during the internal and external quality control.

[Evaluation of umbilical artery resistance index and plasma angiotensin II in pregnancy induced hypertension]. / Evaluarea indicelui de rezistenta al arterei ombilicale si nivelului plasmatic de angiotensina II in hipertensiunea indusa de sarcina.

UNLABELLED: It has already been proven that the renin-angiotensin system has deep implications during the normal pregnancy and along with pregnancy induced hypertension. MATERIAL AND METHOD: We evaluated 14 third trimester pregnancies (8 normotensive and 6 hypertensive women) for body mass index, parity, age, blood pressure (BP), resistance index of the umbilical artery (RIUA), plasma angiotensin II (AngII) level. RESULTS: We identified statistical significant correlations for BP with RIUA, AngII. The covariance analysis showed that the correlation was not influenced by the gestational age. CONCLUSION: AngII and RIUA have statistical significant correlations with BP but no interdependence between them was identified.

[Biological and evolutive spectrum of patients ANCA present]. / Aspecte paraclinice si evolutive la pacientii cu ANCA pozitivi.

AIM: To analyse the immuno-biological profile of patients with antineutrophil cytoplasmic antibodies (ANCA) presents. This is a retrospective study of all ANCA positives cases from Nephrology Clinic Iasi during five years (1998-2003) on a cohort of 97 patients. RESULTS: pANCA was positive in 28, cANCA was positive in 60 and the others had pANCA and cANCA. The diseases ANCA associated were: systemic vasculitis, rapidly progressive glomerulonephritis (GNRP), chronic renal failure of an unknown etiology. ANCA presence was associated with inflammatory syndrome in 83.5%, anemic syndrome in 71.1%. From the patients with renal disfunction (71.1%), half of them presented an increased level of creatinine (>6 mg%). Renal biopsy proven especially crescentic glomerulonephritis. The complications and the diseases were more frequent in cANCA cases. CONCLUSIONS: cANCA presence is a negative prognosis factor in vasculitis; testing ANCA alone cannot be used to guide treatment.

[Retrospective study of 97 ANCA-positive patients: epidemiologic and clinical spectrum]. / Analiza retrospectiva a 97 de cazuri cu ANCA pozitivi: aspecte epidemiologice si clinice.

UNLABELLED: We analysed the clinical profile of antineutrophil cytoplasmic antibodies (ANCA) positive patients in a retrospective study including all cases of ANCA positivity (determined by ELISA) from the Nephrology Clinic, Parhon University Hospital Iasi during the interval 1998-2003. There were 97 ANCA positive patients (mean age 43.7 s18-75t years, female/male ratio 1.55), of whom almost two thirds had c-ANCA, almost one third p-ANCA, while 9 patients had both types of antibodies. The incidence was 22.5/pmp for the North-Eastern province of Romania. Just 19.3% from the suspected cases with ANCA-associated disease were positive for these antibodies. 47.7% had systemic vasculitis (10 with microscopic polyangiitis--MA, 6 with Wegener's granulomatosis--WG, 1 with Churg-Strauss angiitis, 29 with non-specific vasculitis--NSV). Twenty-seven (27.8%) had connective tissue disease--CTD (systemic lupus erythematosus, rheumatoid arthritis, polymyositis, systemic sclerosis, mixed connective tissue disease, and sarcoidosis), while in 5 cases ANCA were associated with other diseases. Nine cases presented with rapid progressive glomerulonephritis (RPGN) without signs of systemic involvement, and other ten with advanced chronic renal failure (CRF). The most frequent clinical manifestations involved the kidney (71%), the skin, the muscles and joints, and the cardiovascular system. CONCLUSIONS: ANCA positivity is associated with a wide spectrum of diseases, mostly with CTD and NSV. c-ANCA was predominantly seen in WG and advanced CRF, while p-ANCA was associated with MA. In nonspecific vasculitis and connective tissue diseases, both patterns were present. We recommend ANCA determination as a screening method in all cases with renal dysfunction and nephritic syndrome and/or with signs of systemic vasculitis and/or collagenosis.