Annual change in the extracellular fluid/intracellular fluid ratio and mortality in patients undergoing maintenance hemodialysis.

We aimed to investigate whether annual change in the extracellular fluid to intracellular fluid (ΔECF/ICF) ratio can accurately predict mortality in hemodialysis patients. Totally, 247 hemodialysis patients were divided into two groups according to the median baseline ECF/ICF ratio of 0.563 and ΔECF/ICF ≥ 0% or < 0% during the first year, respectively. Thereafter, they were divided into four groups according to each cutoff point and were followed up for mortality assessment. The ECF/ICF ratio increased from 0.566 ± 0.177 to 0.595 ± 0.202 in the first year (P = 0.0016). During the 3.4-year median follow-up, 93 patients died (42 cardiovascular-specific causes). The baseline ECF/ICF ≥ 0.563 and ΔECF/ICF ≥ 0% were independently associated with all-cause mortality (adjusted hazard ratio [aHR] 4.55, 95% confidence interval [CI] 2.60-7.98 and aHR 8.11, 95% CI 3.47-18.96, respectively). The aHR for ECF/ICF ≥ 0.563 and ΔECF/ICF ≥ 0% vs. ECF/ICF < 0.563 and ΔECF/ICF < 0% was 73.49 (95% CI 9.45-571.69). For model discrimination, adding the ΔECF/ICF (0.859) alone and both the baseline ECF/ICF and ΔECF/ICF (0.903) to the established risk model (0.746) significantly improved the C-index. Similar results were obtained for cardiovascular mortality. In conclusion, the ΔECF/ICF ratio could not only predict all-cause and cardiovascular mortality but also improve predictability of mortality in hemodialysis patients.

FRET-based assay for intracellular evaluation of α-synuclein aggregation inhibitors.

Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.

Physiological response of the moderately halophilic psychrotolerant strain Chromohalobacter sp. N1 to salinity change and low temperature.

The available information on de novo synthesized compatible solutes in response to high medium salinity by bacteria of the Chromohalobacter genus is limited to studies of the mesophilic moderately halophilic strain Chromohalobacter salexigens DSM 3043T. Therefore, there is a need for studies of representatives of other species of the Chromohalobacter genus of the Halomonadaceae family. A moderately halophilic psychrotolerant bacterium, strain N1, closely related to the species Chromohalobacter japonicus was isolated from the salt crust of a rock salt waste pile in Berezniki, Perm Krai, Russia. An intracellular pool of compatible solutes of strain N1 was investigated by NMR spectroscopy. Cells grown in the presence of 5% NaCl at optimal growth temperature (28 °C) accumulated ectoine, glutamate, N(4)-acetyl-l-2,4-diaminobutyrate (NADA), alanine, trehalose, hydroxyectoine, and valine. Such a combination of compatible solutes is unique and distinguishes the strain from C. salexigens DSM 3043T. Hyperosmotic stress induced by 15% NaCl caused the accumulation of ectoine, NADA, and hydroxyectoine but led to a decrease in the amount of alanine, valine, and trehalose. The intracellular pool of glutamate was not significantly changed. A reduction of the growth temperature from 28 to 5 °C led to an increase in the amount of ectoine, NADA, trehalose, and hydroxyectoine. Ectoine was the major compatible solute.

Correlation between lactate dehydrogenase/pyruvate dehydrogenase activities ratio and tissue pH in the perfused mouse heart: A potential noninvasive indicator of cardiac pH provided by hyperpolarized magnetic resonance.

Cardiovascular diseases account for more than 30% of all deaths worldwide and many could be ameliorated with early diagnosis. Current cardiac imaging modalities can assess blood flow, heart anatomy and mechanical function. However, for early diagnosis and improved treatment, further functional biomarkers are needed. One such functional biomarker could be the myocardium pH. Although tissue pH is already determinable via MR techniques, and has been since the early 1990s, it remains elusive to use practically. The objective of this study was to explore the possibility to evaluate cardiac pH noninvasively, using in-cell enzymatic rates of hyperpolarized [1-13 C]pyruvate metabolism (ie, moles of product produced per unit time) determined directly in real time using magnetic resonance spectroscopy in a perfused mouse heart model. As a gold standard for tissue pH we used 31 P spectroscopy and the chemical shift of the inorganic phosphate (Pi) signal. The nonhomogenous pH distribution of the perfused heart was analyzed using a multi-parametric analysis of this signal, thus taking into account the heterogeneous nature of this characteristic. As opposed to the signal ratio of hyperpolarized [13 C]bicarbonate to [13 CO2 ], which has shown correlation to pH in other studies, we investigated here the ratio of two intracellular enzymatic rates: lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), by way of determining the production rates of [1-13 C]lactate and [13 C]bicarbonate, respectively. The enzyme activities determined here are intracellular, while the pH determined using the Pi signal may contain an extracellular component, which could not be ruled out. Nevertheless, we report a strong correlation between the tissue pH and the LDH/PDH activities ratio. This work may pave the way for using the LDH/PDH activities ratio as an indicator of cardiac intracellular pH in vivo, in an MRI examination.

The sour side of vitamin C might mediate neuroprotective, anticonvulsive and antidepressant-like effects.

In animal experiments, neuroprotective, anticonvulsive and antidepressant-like properties have been increasingly attributed to administrations of ascorbic acid (AA, vitamin C) in at least medium (low millimolar) doses, which however await validation in well controlled clinical studies. In mammalian cortical and subcortical neurons, small to modest acidification (<0.4-0.5 pH-units) is belonging to the key strategies for controlling local excitability and is associated with neuroprotection, e.g. by limiting excitotoxicity. Such acidifications are furthermore involved in the mechanisms of some anticonvulsants and antidepressants. As AA-transport and regulation of intracellular pH (pHi) are closely interwoven on the level of special transmembrane solute carriers, I suppose that the aforementioned beneficial AA-effects might be based upon a discrete "hormetic" acidification of cortical and or subcortical neurons via an AA-mediated weakening of their pHi-regulation. This assumption is supported by findings in non-neuronal cells suggesting both, intracellular acidification and inhibition of a core-element of the pHi-regulation apparatus by millimolar AA. In mammalian subcortical neurons, there is already first evidence of a modest acidification after adding low millimolar AA.

Evidence-based guidelines for controlling pH in mammalian live-cell culture systems.

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid-base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3- and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.

Aging is associated with a mild acidification in neocortical human neurons in vitro.

The intracellular pH (pHi) in the cytosol of mammalian central neurons is tightly regulated and small pHi-fluctuations are deemed to modulate inter-/intracellular signaling, excitability, and synaptic plasticity. The resting pHi of young rodent hippocampal pyramidal neurons is known to decrease alongside aging for about 0.1 pH-units. There is no information about the relationship between age and pHi of human central neurons. We addressed this knowledge gap using 26 neocortical slices from 12 patients (1-56-years-old) who had undergone epilepsy surgery. For fluorometric recordings, the slice-neurons were loaded with the pHi-sensitive dye BCECF-AM. We found that the pyramidal cells' resting pHi (n = 26) descended linearly alongside aging (r = - 0.71, p < 0.001). This negative relationship persisted, when the sample was confined to specific brain regions (i.e., middle temporal gyrus, 23 neurons, r = - 0.68, p < 0.001) or pathologies (i.e., hippocampus sclerosis, 8 neurons, r = - 0.78, p = 0.02). Specifically, neurons (n = 9, pHi 7.25 ± 0.12) from young children (1.5 ± 0.46-years-old) were significantly more alkaline than neurons from adults (n = 17, 38.53 ± 12.38 years old, pHi 7.08 ± 0.07, p < 0.001). Although the samples were from patients with different pathologies the results were in line with those from the rodent hippocampal pyramidal neurons. Like a hormetin, the age-related mild pHi-decrease might contribute to neuroprotection, e.g., via limiting excitotoxicity. On the other hand, aging cortical neurons could become more vulnerable to metabolic overstress by a successive pHi-decrease. Certainly, its impact for the dynamics in short and long-term synaptic plasticity and, ultimately, learning and memory provides a challenge for further research.

Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass.

OBJECTIVE: To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. DESIGN: Prospective study. SETTING: Academic and in vitro fertilization units. PATIENT(S): Sixteen donated cryopreserved embryos at blastocyst stage. INTERVENTION(S): BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). MAIN OUTCOME MEASURE(S): Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. RESULT(S): Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. CONCLUSION(S): BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised.

Clinical Pharmacokinetics and Pharmacodynamics of Drugs in the Central Nervous System.

Despite contributing significantly to the burden of global disease, the translation of new treatment strategies for diseases of the central nervous system (CNS) from animals to humans remains challenging, with a high attrition rate in the development of CNS drugs. The failure of clinical trials for CNS therapies can be partially explained by factors related to pharmacokinetics/pharmacodynamics (PK/PD), such as lack of efficacy or improper selection of the initial dosage. A focused assessment is needed for CNS-acting drugs in first-in-human studies to identify the differences in PK/PD from animal models, as well as to choose the appropriate dose. In this review, we summarize the available literature from human studies on the PK and PD in brain tissue, cerebrospinal fluid, and interstitial fluid for drugs used in the treatment of psychosis, Alzheimer's disease and neuro-HIV, and address critical questions in the field. We also explore newer methods to characterize PK/PD relationships that may lead to more efficient dose selection in CNS drug development.

The metabolism and transport of 1,5-anhydroglucitol in cells.

AIMS: Our previous studies demonstrated that serum 1,5-anhydroglucitol (1,5-AG) levels increased slightly rather than declined after an acute glucose load. Therefore, the current study aims at exploring the transport and metabolic characteristics of 1,5-AG, as well as the effect of glucose on 1,5-AG transport. METHODS: Km and Vmax were determined to measure the affinity of glucose oxidase (GOD) and hexokinase (HK) for 1,5-AG and glucose. HepG2, C2C12, and primary mouse hepatocytes were incubated for 2 h with 1,5-AG at concentrations of 0, 80, and 160 µg/mL. Then, intracellular and extracellular concentrations of 1,5-AG were measured before and after washing with PBS to evaluate the transport and metabolic rates of 1,5-AG. In addition, the influence of an acute glucose load on the transport of 1,5-AG was studied. RESULTS: The affinity of GOD and HK for 1,5-AG is 5 and 42.5% of that for glucose, respectively. Moreover, there is no de novo synthesis of 1,5-AG, and its metabolic rate is < 3%. After a 2 h incubation with additional 1,5-AG, the intracellular levels of 1,5-AG were 50-80% of extracellular levels. Moreover, intracellular 1,5-AG concentrations decreased rapidly and reached zero following the removal of 1,5-AG from the external medium. In addition, an acute glucose load can affect the dynamic balance of 1,5-AG, causing the intracellular 1,5-AG levels to decline significantly and the extracellular levels to increase slightly in HepG2 cells. CONCLUSIONS: Unlike glucose, 1,5-AG is hard to be metabolized in vivo, and its transport is influenced by an acute glucose load in hepatocytes.

Proteasome-mediated degradation of tyrosine hydroxylase triggered by its phosphorylation: a new question as to the intracellular location at which the degradation occurs.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability of TH determined by the degradation remains unknown; although the TH molecule phosphorylated at its Ser19 was observed in the nucleus, and the phosphorylation suspected to trigger its proteasome-mediated degradation. Computer-assisted analysis using the cNLS Mapper program predicted that two sequences of nuclear localization signals (NLS) exist in the N-terminus of TH molecule containing the phosphorylation sites Ser19, Ser31, and Ser40 (Pro9-Arg38 and Lys12-Ile42): the NLS scores indicated that TH could become localized in both nucleus and cytoplasm. Moreover, inhibition of the importin α/ß-mediated nuclear import pathway increased the level of TH phosphorylated at its Ser19 in PC12D cells. The results suggest that TH might be imported to nucleus from cytoplasm to be degraded. Recent studies revealed that proteasomes predominantly exist in the nucleus rather than in the cytoplasm to degrade the nuclear proteins related to cell-cycle progression, gene expression, DNA damage, and DNA repair. Therefore, these studies suggest that the relationship between the phosphorylation and the nuclear localization of the TH molecule should be a matter of focus to understand the mechanism of proteasome-mediated degradation of the enzyme as a first priority.

Soft Matter in Lipid-Protein Interactions.

Membrane lipids and cellular water (soft matter) are becoming increasingly recognized as key determinants of protein structure and function. Their influences can be ascribed to modulation of the bilayer properties or to specific binding and allosteric regulation of protein activity. In this review, we first consider hydrophobic matching of the intramembranous proteolipid boundary to explain the conformational changes and oligomeric states of proteins within the bilayer. Alternatively, membranes can be viewed as complex fluids, whose properties are linked to key biological functions. Critical behavior and nonideal mixing of the lipids have been proposed to explain how raft-like microstructures involving cholesterol affect membrane protein activity. Furthermore, the persistence length for lipid-protein interactions suggests the curvature force field of the membrane comes into play. A flexible surface model describes how curvature and hydrophobic forces lead to the emergence of new protein functional states within the membrane lipid bilayer.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

EPR analysis of extra- and intracellular nitric oxide in liver biopsies.

PURPOSE: To develop an assay that can enable the quantification of intra- and extracellular nitric oxide (NO) levels in liver biopsies without application of potentially harmful exogenous NO traps. THEORY: Electron paramagnetic resonance (EPR) spectroscopy is currently the most appropriate method of measuring NO in biological samples due to the outstanding specificity resulting from the interaction of NO with exogenous NO traps. Because such traps are not allowed in clinical settings, we tested the reliability of endogenous NO traps for the determination of NO levels in blood and liver compartments. METHODS: Rats were injected with 0-8 mg/kg lipopolysaccharide (LPS) to gradually induce a systemic inflammatory response. Specific features of NO-hemoglobin and NO-Fe EPR signals were quantified using a specifically developed calibration procedure. RESULTS: Whereas both NO-hemoglobin (NO-HbLIVER BLOOD ) and NO-Fe (NO-FeLIVER ) complexes were detected in nonperfused liver tissue, only NO-Fe complexes were detected in perfused tissue and only NO-Hb complexes were detected in blood (NO-HbBLOOD ). The NO concentrations increased in the sequence NO-HbBLOOD < NO-FeLIVER < NO-HbLIVER BLOOD (9.4, 18.5, 27.9 nmol/cm3 , respectively at 2.5 mg/kg LPS). The detection limit of the method was 0.61 nmol/cm3 for NO-Hb and 0.52 nmol/cm3 for NO-Fe. CONCLUSION: The assay reported here does not influence natural NO pathways and enables the quantification of NO distribution in two liver compartments using a single liver biopsy. Magn Reson Med 77:2372-2380, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Photobiomodulation by Infrared Diode Laser: Effects on Intracellular Calcium Concentration and Nitric Oxide Production of Paramecium.

In Paramecium, cilia beating is correlated to intracellular calcium concentration ([Ca2+ ]i) and nitric oxide (NO) synthesis. Recent findings affirm that photobiomodulation (PBM) can transiently increase the [Ca2+ ]i in mammalian cells. In this study, we investigated the effect of both 808 and 980 nm diode laser irradiated with flat-top hand-piece on [Ca2+ ]i and NO production of Paramecium primaurelia, to provide basic information for the development of new therapeutic approaches. In the experiments, the laser power in CW varied (0.1; 0.5; 1; and 1.5 W) to generate the following respective fluences: 6.4; 32; 64; and 96 J cm-2 . The 6.4 J cm-2 did not induce PBM if irradiated by both 808 and 980 nm diode laser. Conversely, the 32 J cm-2 fluence had no effect on Paramecium cells if irradiated by the 808 nm laser, while if irradiated by the 980 nm laser induced increment in swimming speed (suggesting an effect on the [Ca2+ ]i, NO production, similar to the 64 J cm-2 with the 808 nm wavelength). The more evident discordance occurred with the 96 J cm-2 fluence, which had the more efficient effect on PBM among the parameters if irradiated with the 808 nm laser and killed the Paramecium cells if irradiated by the 980 nm laser. Lastly, the 980 nm and 64 or 96 J cm-2 were the only parameters to induce a release of stored calcium.

KR-33028, a potent inhibitor of the Na+/H+ exchanger NHE1, suppresses metastatic potential of triple-negative breast cancer cells.

Hyper-activation of the Na+/H+ exchanger NHE1 occurs at the onset of oncogenic transformation and plays a critical role in breast cancer carcinogenesis. Dysregulation of NHE1 activity results in intracellular alkalinization and the acidification of the extracellular tumor microenvironment that promotes metastasis. Hence, the use of chemical inhibitors of NHE1 as chemotherapeutic agents is an alluring prospect. We previously demonstrated that two structurally different NHE1 inhibitors, EMD87580 [(2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine], and HMA [5-(N,N-hexamethylene)-amiloride], were effective as co-adjuvants to potentiate paclitaxel-mediated cytotoxic chemotherapy in triple-negative breast cancer (TNBC) cells. Both these drugs, however, had reduced or minimal anti-cancer effects when used alone. Here, we tested KR-33028 (4-cyano (benzo[b]thiophene-2-carbonyl)guanidine), a potent and selective inhibitor of NHE1, to determine its efficacy in inhibition of metastatic potential of TNBC cells. In highly invasive MDA-MB-231, moderately invasive MDA-MB-468, and lowly invasive Hs578T TNBC cells, KR-33028 considerably reduced rates of cell migration and anchorage-independent colony growth. Invasion of MDA-MB-231 and MDA-MB-468 cells through extracellular matrix was also dramatically decreased in response to KR-33028. We further tested the effect of KR-33028 on MDA-MB-231 cells lacking NHE1 expression (231koNHE1); no differences were observed between untreated control and KR-33028-treated 231koNHE1 cells. Taken together, our results highlight the in vitro efficacy of KR-33028-mediated NHE1 inhibition on limiting cellular functions that are predictive of metastasis in vivo. We suggest that targeting NHE1 in the development of novel chemotherapeutics could be highly effective in combatting triple-negative breast cancer and that KR-33028 is potentially useful in prevention of metastasis.

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.

Self-Assembled Monomethoxy (Polyethylene Glycol)-b-P(D,L-Lactic-co-Glycolic Acid)-b-P(L-Glutamic Acid) Hybrid-Core Nanoparticles for Intracellular pH-Triggered Release of Doxorubicin.

Triblock copolymers, Monomethoxy (Polyethylene glycol)-b-P(D,L-lactic-co-glycolic acid)-b-P(L-glutamic acid) (mPEG-PLGA-PGlu) with different molecular weights, were synthesized and mPEG(5k)-PLGA(20.5k)-PGlu(7.9k) were self-assembled into negatively charged nanoparticles with a hybrid core of PLGA and PGlu, and a stealth PEG shell. Because of electrostatic interaction with the negative hybrid-core, the model drug, doxorubicin (DOX), could be easily loaded into the hybrid-core nanoparticles with a high drug loading of ca. 25%. The hydrophobic interaction provided by PLGA could increase the stability of drug-loaded nanoparticles with no change in particle size for at least 3 days and only minor drug leakage (< 0.5%) in pH7.4 physiological media. Due to protonation of PGlu block in pH5.0 medium, the hybrid-core of these nanoparticles was destroyed, as shown by transmission electron microscopy, and this resulted in an increase in the pH-triggered release of DOX from 38.9% in pH7.4 release medium to 71% in pH5.0 release medium at 24 h. In vitro cytotoxicity testing involving MCF-7 and NCI-H460 cells showed that DOX-loaded nanoparticles were more cytotoxic to both types of cells than free DOX. Time-dependent cellular uptake of the drug-loaded nanoparticles was observed and at least 4 hours was required for rapid internalization through caveolinmediated endocytosis and macropinocytosis by MCF-7 cells into the endosomes where pH-trigged release of DOX from the nanoparticles occurred. The hybrid-core nanoparticles represent a potentially useful therapeutic delivery system for cationic drugs due to their high drug loading, high stability in physiological media and intracellular pH-triggered release.

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.