Real Time In Vivo Measurement of Ascorbate in the Brain Using Carbon Nanotube‐Modified Microelectrodes

A carbon fiber microelectrode modified with a composite film of carbon nanotubes and Nafion was developed for in vivo ascorbate (AA) measurements in brain tissue. The modified‐microelectrodes exhibit an electrocatalytic activity for AA oxidation by shifting the peak potential negatively to ?0.040 V, showing a sensitivity of 37 pA/µM, a detection limit of 2.5 µM, a response time of 0.3 s and don’t respond to several electroactive compounds found in the brain extracellular space. In the rat hippocampus, the basal concentration of AA was 290 µM, and glutamate‐evoked changes in AA were biphasic comprising fast and slow components.     

Evaluation of the Pharmacokinetics of the Pancreastatin Inhibitor PSTi8 Peptide in Rats: Integration of In Vitro and In Vivo Findings

PSTi8 is a pancreastatin inhibitory peptide that is effective in the treatment of diabetic models. This study investigates the pharmacokinetic (PK) properties of PSTi8 in Sprague Dawley rats, for the first time. In vitro and in vivo PK studies were performed to evaluate the solubility, stability in plasma and liver microsomes, plasma protein binding, blood–plasma partitioning, bioavailability, dose proportionality, and gender difference in PK. Samples were analyzed using the validated LC-MS/MS method. The solubility of PSTi8 was found to be 9.30 and 25.75 mg/mL in simulated gastric and intestinal fluids, respectively. The protein binding of PSTi8 was estimated as >69% in rat plasma. PSTi8 showed high stability in rat plasma and liver microsomes and the blood–plasma partitioning was >2. The bioavailability of PSTi8 after intraperitoneal and subcutaneous administration was found to be 95.00 ± 12.15 and 78.47 ± 17.72%, respectively, in rats. PSTi8 showed non-linear PK in dose proportionality studies, and has no gender difference in the PK behavior in rats. The high bioavailability of PSTi8 can be due to high water solubility and plasma protein binding, low clearance and volume of distribution. Our in vitro and in vivo findings support the development of PSTi8 as an antidiabetic agent.     

Poloxamer 407/188 Binary Thermosensitive Gel as a Moxidectin Delivery System: In Vitro Release and In Vivo Evaluation

Moxidectin (MXD) is an antiparasitic drug used extensively in veterinary clinics. In this study, to develop a new formulation of MXD, a thermosensitive gel of MXD (MXD-TG) was prepared based on poloxamer 407/188. Furthermore, the gelation temperature, the stability, in vitro release kinetics and in vivo pharmacokinetics of MXD-TG were evaluated. The results showed that the gelation temperature was approximately 27 °C. MXD-TG was physically stable and can be released continuously for more than 96 h in vitro. The Korsmeyer–Peppas model provided the best fit to the release kinetics, and the release mechanism followed a diffusive erosion style. MXD-TG was released persistently for over 70 days in sheep. Part of pharmacokinetic parameters had a difference in female and male sheep (p < 0.05). It was concluded that MXD-TG had a good stability, and its release followed the characteristics of a diffusive erosion style in vitro and a sustained release pattern in vivo.     

Glucose Biosensor Based on Oxygen Electrode Part IV: In Vivo Evaluation of the Rechargeable Glucose Sensor

Abstract

A glucose monitoring system consisting of a pair of amperometric sensors: a glucose biosensor based on oxygen electrode and an oxygen sensor, two miniature potentiostats, an instrumentation amplifier and a data logger has been developed. The glucose sensor has linear response to the glucose concentration in vitro at 37°C up to 26 mM (480 mg/dL) in the phosphate buffer solution (pH 7.4), and linear range up to 21 mM (380 mg/dL) in undiluted bovine plasma. The system was evaluated in vivo with the sensors subcutaneously implanted in healthy mongrel dogs. During the implantation the system output was continuously recorded. The results of short-term subcutaneous implantation of the integrated system demonstrated good agreement between the glucose concentration measured by the biosensor and that obtained using standard glucose determination methods. The delay-time between the tissue glucose level (measured by the biosensor) and the blood glucose level (obtained by standard methodology) was 3 to 10 minutes. During the chronic implantation the biosensor was refilled in vivo. Rejuvenation of the sensor response after refilling was observed demonstrating the potential of such sensors for long-term implantation.     

In Vivo Antistress Effects of Synthetic Flavonoids in Mice: Behavioral and Biochemical Approach

Natural flavonoids, in addition to some of their synthetic derivatives, are recognized for their remarkable medicinal properties. The present study was designed to investigate the in vitro antioxidant and in vivo antistress effect of synthetic flavonoids (flavones and flavonols) in mice, where stress was induced by injecting acetic acid and physically through swimming immobilization. Among the synthesized flavones (F1–F6) and flavonols (OF1–OF6), the mono para substituted methoxy containing F3 and OF3 exhibited maximum scavenging potential against DPPH (2,2-diphenyl-1-picrylhydrazyl) with IC₅₀ of 31.46 ± 1.46 μg/mL and 25.54 ± 1.21 μg/mL, respectively. Minimum antioxidant potential was observed for F6 and OF6 with IC₅₀ values of 174.24 ± 2.71 μg/mL and 122.33 ± 1.98 μg/mL, respectively, in comparison with tocopherol. The ABTS scavenging activity of all the synthesized flavones and flavonols were significantly higher than observed with DPPH assay, indicating their potency as good antioxidants and the effectiveness of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) assay in evaluating antioxidant potentials of chemical substances. The flavonoids-treated animals showed a significant (* p < 0.05, ** p < 0.01 and *** p < 0.001, n = 8) reduction in the number of writhes and an increase in swimming endurance time. Stressful conditions changed plasma glucose, cholesterol and triglyceride levels, which were used as markers when evaluating stress in animal models. The level of these markers was nearly brought to normal when pre-treated with flavones and flavonols (10 mg/kg) for fifteen days in experimental animals. These compounds also considerably reduced the levels of lipid peroxidation (TBARS: Thiobarbituric acid reactive substances), which was significant (* p < 0.05, ** p < 0.01 and *** p < 0.001, n = 8) compared to the control group. A significant rise in the level of catalase and SOD (super oxide dismutase) was also observed in the treated groups. Diazepam (2 mg/kg) was used as the standard drug. Additionally, the flavonoids markedly altered the weight of the adrenal glands, spleen and brain in stress-induced mice. The findings of the study suggest that these flavonoids could be used as a remedy for stress and are capable of ameliorating diverse physiological and biochemical alterations associated with stressful conditions. However, further experiments are needed to confirm the observed potentials in other animal models, especially in those with a closer resemblance to humans. Toxicological evaluations are also equally important.     

中红外光纤技术用于口腔肿瘤在体原位检测的研究

肿瘤是严重威胁人类健康和生命的疾病,早期诊断和及时治疗是提高肿瘤病人存活率的重要因素,肿瘤的发生和发展一般可分为3个阶段：(1)基因突变;(2)生物分子组成和结构发生改变;(3)细胞和组织形态发生变化,目前常用的影像学方法只能检测较大的肿块,组织标本的病理诊断法需在     

Evaluation of 2-Thioxoimadazolidin-4-one Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC₅₀ values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC₅₀ values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.     

Passive Tumor Targeting of Polymer Therapeutics: In Vivo Imaging of Both the Polymer Carrier and the Enzymatically Cleavable Drug Model

The enzymatic release of a model drug from a polymer carrier inside a tumor using multispectral optical imaging in vivo in nude mice bearing colorectal carcinomas HT‐29 and DLD‐1 is demonstrated. Much higher release rate in vivo from a linear (30 kDa) (N‐2‐hydroxypropyl)methacrylamide‐based polymer compared with a high molecular weight branched (170 kDa) polymer conjugate is observed, probably due to steric hindrance of the cleavable spacer of the latter polymer to proteolytic enzymes. There is no significant difference in the relative biodistribution of the two polymers, but the branched polymer circulates much longer. Both polymers are labeled with two different fluorophores. Dyomics‐676 as a drug model is attached to the polymer via an enzymatically cleavable Gly‐Phe‐Leu‐Gly spacer; Dyomics 782 is bound to the same polymer via a nondegradable amide bond, enabling the tracking of the polymer carrier after i.v. application to mice.

     

中性红掺杂SiO2纳米粒子修饰的酶阵列传感器同时测定葡萄糖、乳酸和L-谷氨酸的研究

本文以中性红为核，二氧化硅为壳，利用反相微乳液技术，通过正硅酸四乙酯的水解制备了掺杂有中性红的二氧化硅纳米粒子，并用TEM技术进行了表征。核中性红能够催化测定葡萄糖，乳酸和L-谷氨酸的反应，而壳二氧化硅不仅克服了电活性物质中性红易流失的缺点，且具有高的生物亲和性。分别与葡萄糖氧化酶、乳酸氧化酶以及L-谷氨酸氧化酶混合后，修饰在碳阵列电极表面。最后在该酶阵列电极表面滴加一层Nafion, 防止电活性物质抗坏血酸、尿酸等的干扰。该酶阵列传感器与流动注射分析技术(FIA)相结合，可应用于同时检测大鼠血样中的葡萄糖，乳酸和L-谷氨酸浓度。该方法无需通过传统的色谱柱的分离，大大简化了实验条件，为这一领域的研究提供了有效的分析方法。     

In Vivo Anticancer Evaluation of 6b,a Non-Covalent Imidazo[1,2-a]quinoxaline-Based Epidermal Growth Factor Receptor Inhibitor against Human Xenograft Tumor in Nude Mice

Tyrosine kinase inhibitors are validated therapeutic agents against EGFR-mutated non-small cell lung cancer (NSCLC). However, the associated critical side effects of these agents are inevitable, demanding more specific and efficient targeting agents. Recently, we have developed and reported a non-covalent imidazo[1,2-a]quinoxaline-based EGFR inhibitor (6b), which showed promising inhibitory activity against the gefitinib-resistant H1975(L858R/T790M) lung cancer cell line. In the present study, we further explored the 6b compound in vivo by employing the A549-induced xenograft model in nude mice. The results indicate that the administration of the 6b compound significantly abolished the growth of the tumor in the A549 xenograft nude mice. Whereas the control mice bearing tumors displayed a declining trend in the survival curve, treatment with the 6b compound improved the survival profile of mice. Moreover, the histological examination showed the cancer cell cytotoxicity of the 6b compound was characterized by cytoplasmic destruction observed in the stained section of the tumor tissues of treated mice. The immunoblotting and qPCR results further signified that 6b inhibited EGFR in tissue samples and consequently altered the downstream pathways mediated by EGFR, leading to a reduction in cancer growth. Therefore, the in vivo findings were in corroboration with the in vitro results, suggesting that 6b possessed potential anticancer activity against EGFR-dependent lung cancer. 6b also exhibited good stability in human and mouse liver microsomes.     

A Single Biosensor for Evaluating the Levels of Copper Ion and L‐Cysteine in a Live Rat Brain with Alzheimer's Disease

Copper ion (Cu²⁺) and L ‐cysteine (CySH) are closely correlated with physiological and pathological events of Alzheimer’s Disease (AD), however the detailed mechanism is still unclear, mainly owing to a lack of accurate analytical methods in live brains. Herein, we report a single biosensor for electrochemical ratiometric detection of Cu²⁺ and CySH in live rat brains with AD. N,N‐di‐(2‐picoly)ethylenediamine (DPEA) is first synthesized for specific recognition of Cu²⁺ to form a DPEA–Cu²⁺ complex. This complex shows high selectivity for CySH owing to the release of Cu²⁺ from the complex through CySH binding to Cu²⁺ center. In parallel, 5′‐MB‐GGCGCGATTTTTTTTTTTTT‐SH‐3′ (HS‐DNA‐MB, MB=Methylene Blue) is designed as an inner‐reference for providing a built‐in correction to improve the accuracy. As a result, combined with the amplified effect of Au nanoleaves, our single ratiometric biosensor can be successfully applied in real‐time detection of Cu²⁺ and CySH in the live rat brains with AD. To our knowledge, this is the first report on the accurate concentrations of Cu²⁺ and CySH in live rat brains with AD.     

An Electrochemophysiological Microarray for Real-Time Monitoring and Quantification of Multiple Ions in the Brain of a Freely Moving Rat

Herein, we present an electrochemophysiological microarray (ECPM) for real-time mapping and simultaneous quantification of chemical signals for multiple ions in the deep brain of a freely moving rat, in which microelectrode arrays were developed for direct determination of multiple ions using open-circuit potentiometry. Specific recognition ionophores were synthesized and optimized for determination of K⁺, Ca²⁺, Na⁺ and pH. A reference electrode was also developed to avoid interferences in the brain. The microarrays were successfully applied in real-time monitoring and quantification of ions in a live brain. The extra current-free potentiometry allowed mapping and biosensing of chemical signals, together with recording of electrical signals in the whole brain without cross-talk, for the first time. Furthermore, the ECPM provided a platform for real-time monitoring of the dynamic changes of multiple ions in the deep brain of freely moving rat during a seizure.     

Development of an Efficient Biosensor for the In Vivo Monitoring of Cu+ and pH in the Brain: Rational Design and Synthesis of Recognition Molecules

An efficient biosensor was created for the ratiometric monitoring of Cu⁺ and pH in the brain using both current and potential outputs. A series of N ,N ‐bis(2‐[2‐(ethylthio)ethyl])‐based (NS4s) derivatives was designed for the specific recognition of Cu⁺. After systematically evaluating the electrochemical parameters of Cu⁺ oxidation by tuning alkyl chain length, polyaromatic structure, and substitute group site of NS4, N ,N ‐bis(2‐[2‐(ethylthio)ethyl])‐2‐naphthamide (NS4‐C1) was finally optimized for Cu⁺ detection as it showed the most negative potential and the largest current density. At the same time, 9,10‐anthraquinone was used as a selective pH sensor with 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) as an internal reference. This single biosensor with both current and potential signal outputs can simultaneously determine Cu⁺ concentrations from 0.5 to 9.5 μm and pH values ranging from 6.0 to 8.0. The efficient biosensor was applied to the simultaneous detection of Cu⁺ and pH in the live brain. The average levels of Cu⁺ were reported for the first time in the cortex, hippocampus, and striatum in a mouse model of Alzheimer's disease.     

In Vivo Monitoring of the Thiols in Rat Striatum by Liquid Chromatography with Amperometric Detection at a Functionalized Multi‐Wall Carbon Nanotubes Modified Electrode

A new chemically modified electrode (CME) was fabricated, which was based on the immobilization of multi‐wall carbon nanotubes fuctionalized with carboxylic group (MWNT‐COOH). The results indicated that the CME exhibited efficiently electrocatalytic oxidation for L ‐cysteine and glutathione with relatively high sensitivity, stability and long‐life. Coupled with HPLC, the MWNT‐COOH CME was utilized for amperometric detection of the thiols. The peak currents of L ‐cysteine and glutathione were linear to their concentrations ranging from 3.0×10^?7 to 1.0×10^?3 mol/L with the calculated detection limit (S/N=3) of 1.2×10^?7, 2.2×10^?7 mol/L, respectively. The method had been successfully applied to assess the contents of L ‐cysteine and glutathione in rat striatal microdialysates.     

An Electrochemophysiological Microarray for Real‐Time Monitoring and Quantification of Multiple Ions in the Brain of a Freely Moving Rat

Herein, we present an electrochemophysiological microarray (ECPM) for real‐time mapping and simultaneous quantification of chemical signals for multiple ions in the deep brain of a freely moving rat, in which microelectrode arrays were developed for direct determination of multiple ions using open‐circuit potentiometry. Specific recognition ionophores were synthesized and optimized for determination of K⁺, Ca²⁺, Na⁺ and pH. A reference electrode was also developed to avoid interferences in the brain. The microarrays were successfully applied in real‐time monitoring and quantification of ions in a live brain. The extra current‐free potentiometry allowed mapping and biosensing of chemical signals, together with recording of electrical signals in the whole brain without cross‐talk, for the first time. Furthermore, the ECPM provided a platform for real‐time monitoring of the dynamic changes of multiple ions in the deep brain of freely moving rat during a seizure.     

Preclinical Assessment Addressing Intravenous Administration of a [68Ga]Ga-PSMA-617 Microemulsion: Acute In Vivo Toxicity,Tolerability, PET Imaging,and Biodistribution

It has been herein presented that a microemulsion, known to be an effective and safe drug delivery system following intravenous administration, can be loaded with traces of [⁶⁸Ga]Ga-PSMA-617 without losing its properties or causing toxicity. Following tolerated IV injections the capability of the microemulsion in altering [⁶⁸Ga]Ga-PSMA-617 distribution was presented at 120 min post injection based on its ex vivo biodistribution results.     

Bridging the gap between metabolic profile determination and visualization in neurometabolic disorders: a multivariate analysis of proton magnetic resonance in vivo spectra

The purpose of this work was to check the degree of overlap between rare inborn errors of metabolism and other neurological disorders using principal component analysis of proton magnetic resonance spectroscopy (¹H MRS) in vivo data. We examined 60 patients (median age of 22 months). Fourteen of them were diagnosed with neurometabolic disorders (three cases of metachromatic leukodystrophy, two cases of Canavan disease, two cases of megalencephalic leukoencephalopathy with subcortical cysts, three cases of mitochondrial cytopathy, one case of nonketotic hyperglycinemia, one case of globoid leukodystrophy, one case of congenital disorders of glycosylation, and one case of ethylmalonic encephalopathy). The remaining 46 patients were diagnosed with epilepsy, cerebral palsy, and developmental delay. Results obtained from principal component analysis of complete unresolved ¹H MRS in vivo spectra were interpreted parallelly with LCModel‐derived metabolite levels. The main attention was paid to the following metabolites: N‐acetylaspartate, glutamate + glutamine, creatine, choline, myo‐inositol signal with an uncertain contribution of glycine, and glucose. ¹H MRS in vivo coupled with multivariate analysis is an efficient tool in visualization of metabolic abnormalities in several inborn errors of metabolism (metachromatic leukodystrophy, globoid leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts, and Canavan disease). Copyright © 2013 John Wiley & Sons, Ltd.     

Monitoring the Phosphorylation of Phenol Derivatives with Diethyl Chlorophosphate in Liquid–Liquid and Solid–Liquid Phase by In Situ Fourier Transform Infrared Spectroscopy,Part II

The reaction of 4-chlorophenol and 4-nitrophenol with diethyl chlorophosphate carried out in a liquid–liquid and solid–liquid two phase system, respectively, was monitored by in situ Fourier transform IR spectroscopy.     

Triazine Dendrimers for Drug Delivery: Evaluation of Solubilization Properties,Activity in Cell Culture,and In Vivo Toxicity of a Candidate Vehicle

Three criteria are evaluated to assess the potential of a dendrimer based on triazines, 1, for use as a vehicle for drug delivery. These criteria are: (1) its ability to solubilize small hydrophobic guests as measured spectrophotometrically; (2) its ability to deliver a drug in vitro as evaluated using a gene reporter assay; and (3) its in vivo toxicity in mice as determined by autopsy and screens of liver and kidney function. Vehicle 1 solubilizes pyrene to a similar extent to dendrimers based on poly(arylether)s, 4, encapsulating approximately 0.2 molecules of pyrene per dendrimer. This activity is approximately 10-fold greater than that of the more polar poly(propyleneimine) and poly(amidoamine) dendrimers, 2 and 3. Gas-phase computational models reveal that both 1 and 4 have cores that are accessible to solvent, suggesting that these dendrimers can occupy much greater volumes than 2 and 3 whose cores are confined toward the interior of the structure. Electrostatic potential maps can be used to rationalize differences in solubilization between 1 and 4. Precipitation results from mixing cationic 1 with the anionic indomethacin, but not with methotrexate, suggesting that the composition of the drug may dictate the scope of delivery applications. Dendrimer 1 solubilizes 10-hydroxycamptothecin and a novel bisindolemethane; approximately four and five molecules of drug per dendrimer are solubilized, respectively. In cell-culture experiments using a luciferase reporter gene assay, the dendrimer:bisindolemethane conjugate shows comparable activity to the bisindolemethane delivered in aqueous DMSO, suggesting that the dendrimer does not preclude delivery of the molecule to an intracellular target. Preliminary toxicology studies of 1 in mice show that this molecule has no adverse toxicity to the kidneys or the liver in single doses delivered intraperitoneally up to 10?mg/kg.