DNA adductomics aided rapid screening of genotoxic impurities using nucleosides and 3D bioprinted human liver organoids.

Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.

Simultaneous determination of three strong polarity herbicides in tea by ion chromatography-triple quadrupole mass spectrometry.

Due to lack of chromogenic groups and fluorescence groups, high boiling point, high polarity, low volatility, and small molecular weight of glyphosate, glufosinate and bentazone, the detection of three analyses were limited in all kinds of food. Herein, a method for the simultaneous determination of glyphosate, glufosinate and bentazone in tea by ion chromatography tandem triple quadrupole mass spectrometry (IC-MS) was developed, which is without organic solvent and complex derivatization. The recoveries of three compounds in different teas (black tea, green tea, white tea) ranged from 80.40 % to 107.00 %, and the intraday precision (n = 6) ranged from 0.57 % to 9.90 %, the daytime precision ranged from 1.00 % to 5.30 %, the quantitative limit (LOQ) ranged from 0.36 to 1.30 µg/L, and the detection limit (LOD) ranged from 0.11 to 0.39 µg/L. Furthermore, the detection limit and quantitative limit of glyphosate, glufosinate and bentazone by this method are lower than other methods in real samples. Meanwhile, the established method was successfully applied to determine the terminal residues of the three analytes in twelve tea samples from commercial market. Therefore, this method can provide reliable technical support for the study of residue status in vegetables and fruits.

Solubilisation and Enhanced Oral Absorption of Curcumin Using a Natural Non-Nutritive Sweetener Mogroside V.

Background: Curcumin (CUR) is a functional ingredient from the spice turmeric. It has attracted considerable attention recently, owing to its diverse biological activities. However, curcumin has low water solubility, which limited its applications. Some sugar molecules were found to be able to solubilise poorly water-soluble compounds by forming micelles in aqueous solutions. Purpose: To improve the water solubility and oral absorption of CUR, using a non-nutritive natural sweetener, namely, Mogroside V (Mog-V). Methods: A solid dispersion of CUR in Mog-V was prepared using a solvent evaporation method. The solid dispersion was characterised by using X-ray diffraction and differential scanning calorimetry. The solid dispersion can dissolve in water to form micelles with a diameter of ~160 nm, which were characterised by using dynamic light scattering. To find out the mechanism of solubilisation, the aggregation behaviour of Mog-V molecules in aqueous solution was investigated using nuclear magnetic resonance spectroscopy. Finally, oral absorption of CUR in the solid dispersion was evaluated using a rodent model. Results: A solid dispersion was formed in a ratio of 1 CUR to 10 Mog-V by weight. Upon dissolution into water, CUR laden micelles formed via self-assembly of Mog-V molecules, which increased the solubility of CUR by nearly 6000 times compared with pure CUR crystals. In rats, the solid dispersion increased the oral absorption of CUR by 29 folds, compared with CUR crystals. In terms of solubilisation mechanism, it was found that Mog-V self-assembled into micelles with a core-shell structure and CUR molecules were incorporated into the hydrophobic core of the Mog-V micelles. Conclusion: Mog-V can form a solid dispersion with CUR. Upon dissolution in water, the Mog-V in the solid dispersion can self-assemble into micelles, which solubilise CUR and increase its oral absorption.

A novel sandwich impedimetric immunosensor for detection of apolipoprotein-A1 based on the gold nanoparticle-hybridized mercapto-ß-cyclodextrin-Pb(II) metal-organic framework.

A novel sandwich electrochemical impedimetric immunosensor was proposed to detect apolipoprotein-A1 (Apo-A1), a common biomarker for bladder cancer. The molybdenum disulfide/graphene quantum dot (MoS2/GQD) nanocomposites were modified on the surface of a glassy carbon electrode (GCE) and used to immobilize the biotinylated antibody (Ab1) with the help of chitosan and glutaraldehyde (denoted as BSA/Ab1/CHIT/MoS2/GQD/GCE). Pb(II)-thiol-ß-cyclodextrin metal-organic framework (denoted as Pb-MOF) was synthesized with lead metal ions and thiol-ß-cyclodextrin ligands by a one-pot solvothermal method, and then, gold nanoparticles were modified on the surface of Pb-MOF (Pb-MOF-AuNPs) by Au-S bond, which was used as signal label for the recombinant antibody (Ab2). When the immunosensor of BSA/Ab1/CHIT/MoS2/GQD/GCE reacted with Apo-A1, Pb-MOF-AuNPs-Ab2/BSA was connected to the electrode when immunoreaction occurred, and an immune sandwich structure was formed, which led to significantly increased charge transfer resistance of electrochemical probe for ferrocyanide (II)/(III) within the frequency range 10-1 ~ 105 Hz at 5 mV amplitude and the potential of 0.180 V (vs. SCE). Based on this principle, the quantitative detection of Apo-A1 was established. The relative change of electrochemical resistance and the logarithmic value of Apo-A1 concentration showed a linear relationship with a linear coefficient of 0.9989 in the range 1.00 pg mL-1 and 1.00 µg mL-1 with the limit of detection of 0.30 pg mL-1. The selectivity, repeatability, and other performance of the proposed immunosensor were also investigated. The immunosensor was successfully applied to the detection of real serum and urine samples with recovery in the range 96.4 ~ 109.1% (RSD < 3.8%), indicating that it could be helpful for the clinical diagnosis of bladder cancer.

The Biocomplex Assembled from Antigen Peptide and Toll-like Receptor Agonist Improved the Immunity against Pancreatic Adenocarcinoma In Vivo.

Purpose: One of the biggest challenges in cancer immunotherapy is generating robust cancer-specific immunity. This work describes using a biocomplex assembled from a toll-like receptor agonist CpG oligodeoxynucleotide 1826 (CpG) and a pancreatic cancer antigen peptide mesothelin for tuning pancreatic tumor immunity. Methods: This biocomplex was assembled via electrostatic interactions and characterized in size, morphology, zeta potential, and cargo loading. The effect of biocomplex on cell viability and activation of DCs and macrophages were measured by flow cytometry. The production of cytokines (GM-CSF, TNF, and IL-6) was evaluated by using ELISA kits. The effect of biocomplex on tumor cell proliferation was also evaluated by in vivo tumor model. Result: We can modulate the surface charge of the biocomplex by simply varying the ratios of the two components. In cell models, this biocomplex did not impact cell viability in the antigen-presenting cell (i.e., dendritic cell and macrophage)-directed immunity. Moreover, this biocomplex regulated the secretion of tumor-related cytokines (i.e., GM-CSF, TNF, and IL-6) and promoted the activation of immune cell surface markers (i.e., CD80+, CD86+, and CD40+). In the mouse model, the biocomplex inhibited the tumor burden effectively and promoted the production of effector cytokines. Conclusion: The present studies showed that the biocomplex with antigen peptide and toll-like receptor agonist was able to potentiate the antitumor immunity in vivo. This study will help understanding of immunity in pancreatic cancer and developing new immune therapeutic strategies for pancreatic adenocarcinoma.

Enhancement of Skin Delivery of Drugs Using Proposome Depends on Drug Lipophilicity.

The study aims to investigate the propylene glycol-based liposomes named 'proposomes' in enhancing skin permeation of drugs with different physicochemical properties. Ibuprofen, tofacitinib citrate, rhodamine B, and lidocaine were loaded into proposomes. These drug formulations were analyzed for particle size, zeta potential, polydispersity index, entrapment efficiency, and in vitro skin permeation. The confocal laser scanning microscopy was performed on skin treated with calcein and rhodamine B laden proposomes. The transdermal delivery relative to physicochemical properties of drugs such as logP, melting point, molecular weight, solubility, etc., were analyzed. We tested the safety of the proposomes using reconstructed human skin tissue equivalents, which were fabricated in-house. We also used human cadaver skin samples as a control. The proposomes had an average diameter of 128 to 148 nm. The drug's entrapment efficiencies were in the range of 42.9-52.7%, translating into the significant enhancement of drug permeation through the skin. The enhancement ratio was 1.4 to 4.0, and linearly correlated to logP, molecular weight, and melting point. Confocal imaging also showed higher skin permeation of calcein and rhodamine B in proposome than in solution. The proposome was found safe for skin application. The enhancement of skin delivery of drugs through proposomes was dependent on the lipophilicity of the drug. The entrapment efficiency was positively correlated with logP of the drug, which led to high drug absorption.

Identification and direct determination of fatty acids profile in oleic acid by HPLC-CAD and MS-IT-TOF.

Oleic acid is a pharmaceutical excipient and has been widely used in many dosage forms. It remains unclear in terms of the fatty acids (FAs) profile. In this study, a sensitive and direct method based on high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) was developed to study the compositions of oleic acid. The chromatographic conditions were optimized to achieve good separation and high sensitivity. The components of oleic acid were identified by ion trap/time of flight mass spectrometry (MS-IT-TOF). Twenty-seven FAs were identified based on the exact mass-to-charge ratio and fragments, among which 13 FAs were confirmed with the reference standards. Nine FAs in the oleic acid samples including oleic acid, linolenic acid, myristic acid, palmitoleic acid, linoleic acid, palmitic acid, stearic acid, arachidic acid and behenic acid were simultaneously determined by the developed HPLC-CAD, which showed good linearity with r2>0.999. The limit of detection (LOD) and limit of quantification (LOQ) of 9 FAs were 0.006-0.1 µg mL-1 and 0.032-0.22 µg mL-1, respectively. The components with concentration level not less than 0.03 % (referring to the sample concentration of 1.0 mg mL-1) can be quantified. The mean recovery values of 9 FAs ranged from 96.5%-103.6% at three concentration levels of 80 %, 100 % and 120 %. The repeatability and intermediate precision were less than 5.0 % for oleic acid and components with concentration levels more than 0.05 %. In contrast to the conventional pre-column derivatization gas chromatography (GC), HPLC-CAD could unbiasedly and directly detect more components, especially the FAs with long carbon chains. Overall, the developed novel HPLC-CAD method can ameliorate the deficiency of the indirect GC method recorded in current pharmacopeias, thus having great potential for the comprehensive understanding and quality control of oleic acid.

Enhanced skin retention and permeation of a novel peptide via structural modification, chemical enhancement, and microneedles.

Hyperpigmentation is a common skin condition with serious psychosocial consequences. Decapeptide-12, a novel peptide, has been found to be safer than hydroquinone in reducing melanin content, with efficacy up to more than 50% upon 16 weeks of twice-daily treatment. However, the peptide suffers from limited transcutaneous penetration due to its hydrophilicity and high molecular weight. Therefore, decapeptide-12 was modified by adding a palmitate chain in an attempt to overcome this limitation. Molecular docking results showed that the two peptides exhibited similar biological activity towards tyrosinase. We also tested the effect of chemical penetration enhancers and microneedles to deliver the two peptides into and through skin, using an in vitro human skin permeation method. It was shown that the palm-peptide achieved the best skin retention owing to the increased lipophilicity. In addition, skin permeation of the palm-peptides was enhanced by the chemical skin penetration enhancers, namely, oleic acid and menthol. Skin permeation of the native peptide was enhanced by the microneedle patch but not the chemical skin penetration enhancers. Cutaneous absorption of the palm-peptides was estimated to have achieved its therapeutic concentration within skin. The combinatory approach of using molecular modification, chemical penetration enhancement, and microneedle patch proves to be useful to enhanceskin permeation of the peptides.

Effect of Pharmaceutical Excipients on Intestinal Absorption of Metformin via Organic Cation-Selective Transporters.

Growing evidence has shown that some pharmaceutical excipients can act on drug transporters. The present study was aimed at investigating the effects of 13 commonly used excipients on the intestinal absorption of metformin (MTF) and the underlying mechanisms using Caco-2 cells and an ex vivo mouse non-everted gut sac model. First, the uptake of MTF in Caco-2 cells was markedly inhibited by nonionic excipients including Solutol HS 15, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and crospovidone. Second, transport profile studies showed that MTF was taken up via multiple cation-selective transporters, among which a novel pyrilamine-sensitive proton-coupled organic cation (H+/OC+) antiporter played a key role. Third, Solutol HS 15, polysorbate 40, and polysorbate 60 showed cis-inhibitory effects on the uptake of either pyrilamine (prototypical substrate of the pyrilamine-sensitive H+/OC+ antiporter) or 1-methyl-4-phenylpyridinium (substrate of traditional cation-selective transporters including OCTs, MATEs, PMAT, SERT, and THTR-2), indicating that their suppression on MTF uptake is due to the synergistic inhibition toward multiple influx transporters. Finally, the pH-dependent mouse intestinal absorption of MTF was significantly decreased by Solutol HS 15, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and pyrilamine. In conclusion, this study revealed that a novel transport process mediated by the pyrilamine-sensitive H+/OC+ antiporter contributes to the intestinal absorption of MTF in conjunction with the traditional cation-selective transporters. Mechanistic understanding of the interaction of excipients with cation-selective transporters can improve the formulation design and clinical application of cationic drugs.

Mahuang Decoction Antagonizes Acute Liver Failure via Modulating Tricarboxylic Acid Cycle and Amino Acids Metabolism.

Acute liver failure (ALF) is a serious clinical disorder with high fatality rates. Mahuang decoction (MHD), a well-known traditional Chinese medicine, has multiple pharmacological effects, such as anti-inflammation, anti-allergy, anti-asthma, and anti-hyperglycemia. In this study, we investigated the protective effect of MHD against ALF. In the lipopolysaccharide and D-galactosamine (LPS/D-GalN)-induced ALF mouse model, the elevated activities of the serum alanine and aspartate transaminases as well as the liver pathological damage were markedly alleviated by MHD. Subsequently, a metabolomics study based on the ultrahigh performance liquid chromatograph coupled with Q Exactive Orbitrap mass spectrometry was carried to clarify the therapeutic mechanisms of MHD against ALF. A total of 36 metabolites contributing to LPS/D-GalN-induced ALF were identified in the serum samples, among which the abnormalities of 27 metabolites were ameliorated by MHD. The analysis of metabolic pathways revealed that the therapeutic effects of MHD are likely due to the modulation of the metabolic disorders of tricarboxylic acid (TCA) cycle, retinol metabolism, tryptophan metabolism, arginine and proline metabolism, nicotinate and nicotinamide metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan synthesis, as well as cysteine and methionine metabolism. This study demonstrated for the first time that MHD exerted an obvious protective effect against ALF mainly through the regulation of TCA cycle and amino acid metabolism, highlighting the importance of metabolomics to investigate the drug-targeted metabolic pathways.

Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Thin platelet-like COF nanocomposites for blood brain barrier transport and inhibition of brain metastasis from renal cancer.

Effective treatment of brain metastases is hindered by the blood-brain barrier (BBB) and the rapid development of resistance to drug therapy. Moreover, the clinical application of general formulations is hampered by biological barriers and biological elimination. To tackle this challenge, we report a feasible approach for the assembly of polymer-covalent organic framework (COF) nanocomposites into 150 nm thin platelets as a drug delivery vehicle for enhanced retention in brain tumours. Using intravital imaging, we demonstrate that these polymer-COF nanocomposites are able to traverse the BBB in mice and achieve direct tumour accumulation in intracranial orthotopic models of brain metastasis from renal cancer (BMRC). These nanocomposites can target brain tumour cells and respond to tumour microenvironmental characteristics, including acidic and redox conditions. Intracranial tumour acidity triggers the breakdown of the nanoassemblies to polymer-COF nanocomposites due to the presence of borate bonds. Furthermore, in vivo studies on the nanocomposites showed enhanced brain tumour-targeting efficiency and therapeutic effects compared to those of free-drug dosing. Mice treated with drug-loaded polymer-COF nanocomposites also show protection from systemic drug toxicity and improved survival, demonstrating the preclinical potential of this nanoscale platform to deliver novel combination therapies to BMRC and other central nervous system (CNS) tumours.

Evaluation of Mogroside V as a Promising Carrier in Drug Delivery: Improving the Bioavailability and Liver Distribution of Silybin.

The objective of this work was to investigate the capacity of mogroside V (MOG-V), a food additive, as a novel carrier to improve the bioavailability and liver distribution of silybin (SLY). Solid dispersion particles (SDPs) of SLY/MOG-V were prepared utilizing the solvent evaporation method. The physicochemical characterizations of SDPs were evaluated by using dynamic light scattering (DLS), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) measurements. DLS results demonstrated the formation of nanoparticles (206 nm) of SDPs in water. DSC and PXRD analysis revealed that SLY was in amorphous form or molecularly dispersed in SDPs. SDPs also exhibited a major increase in both dissolution rate and saturation solubility, as evidenced by a 1931-fold improvement (2201 µg/mL) in solubility compared with pure SLY (1.14 µg/mL). The pharmacokinetic study in rats showed that oral absorption of SLY/MOG-V SDPs was dramatically increased. The mean value of AUC until 12 h for SLY/MOG-V SDPs (27,481 ng·min/mL) was 24.5-fold higher than that of pure SLY (1122 ng·min/mL). In vivo tissue distribution experiment in mice confirmed that the major distribution tissue was changed from lungs to liver after SLY was loaded into MOG-V. In addition, even orally administrated to mice at a high dose (4.2 g/kg), MOG-V exhibited no undesirable effect on the plasma glucose concentrations. Thus, MOG-V may have the applicability to serve as an ideal excipient for solubilization or as a novel liver targeting carrier for the delivery of SLY.

A proton-coupled organic cation antiporter is involved in the blood-brain barrier transport of Aconitum alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. AIM OF THE STUDY: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). MATERIALS AND METHODS: The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. RESULTS: In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. CONCLUSIONS: A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.

Keratinocytes maintain compartmentalization between dermal papilla and fibroblasts in 3D heterotypic tri-cultures.

OBJECTIVES: Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells. MATERIALS AND METHODS: Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. RESULTS: The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. CONCLUSIONS: Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.

A miniaturized device for biomembrane permeation analysis.

Transdermal drug delivery is widely investigated as an alternative drug administration route to oral delivery and hypodermic injections. Owing to the availability of human skin samples, in vitro tests are used to predict the in vivo delivery of transdermal drugs. The most widely used validation method is skin permeation using diffusion cells. Traditional diffusion cells, however, are capacious and often require large amounts of skin sample and drugs, which is undesirable, given the scarcity of new drug entities and the limitation of skin sample supply. In this study, we fabricated miniaturized multichannel devices (MCDs) by 3D printing, to minimize the use of skin and drug samples. The MCDs were compared with conventional static diffusion cells and achieved comparable drug permeation profiles. The finite element method-based simulation revealed the efficient carry-off of permeated ingredients by the multichannel devices, and a critical role of distance between the buffer stream and skin sample in determining the flow velocity inside the chamber. The results support these devices as qualified alternatives to Franz cells for in vitro permeation studies using biomembranes, with reduced use of skin and drug samples.

Drug-delivering-drug approach-based codelivery of paclitaxel and disulfiram for treating multidrug-resistant cancer.

Multidrug resistance (MDR) is a common intractable barrier in success of clinical cancer chemotherapy. Codelivery of two drugs using nanocarriers is a commonly used approach to treat the MDR cancer. However, the drug payload in the conventional nanocarriers is low and thus compromises the treatment outcomes. Disulfiram (DSF) is promising to reverse MDR and increases the sensitivity of cancer cells to chemotherapy. While, paclitaxel (PTX) is one of the frequently used anticancer drug. Here, by using a drug-delivering-drug (DDD) strategy based on nanocrystals, hybrid PTX-DSF nanocrystals (PTX-DSF Ns) were developed for codelivery of PTX and DSF to reverse MDR in cancer. The 160-nm PTX-DSF Ns with rod-like morphology had drug-loading up to 43% at mass ratio of 5:1. Interestingly, the nanoparticles entered cells via caveolar endocytosis. By reducing intracellular ATP level and GST activity, PTX-DSF Ns killed the Taxol resistant A549 cells with higher efficiency than PTX alone, exhibiting as 6-fold increase of apoptosis in MDR tumor. The nanoparticles circulated in blood over time, accumulated in tumor efficiently and reduced the tumor volume by 12-fold in MDR tumor-bearing BALB/c nude mice and allowed 12-fold apoptosis in tumor. Additionally, the immunohistochemical examination demonstrated the safety of the nanoparticles. Overall, the DDD strategy-based PTX-DSF Ns have promising potential for the treatment of MDR cancer.

Age-Related Alterations in Electroencephalography Connectivity and Network Topology During n-Back Working Memory Task.

The study of the healthy brain in elders, especially age-associated alterations in cognition, is important to understand the deficits created by Alzheimer's disease (AD), which imposes a tremendous burden on individuals, families, and society. Although, the changes in synaptic connectivity and reorganization of brain networks that accompany aging are gradually becoming understood, little is known about how normal aging affects brain inter-regional synchronization and functional networks when items are held in working memory (WM). According to the classic Sternberg WM paradigm, we recorded multichannel electroencephalography (EEG) from healthy adults (young and senior) in three different conditions, i.e., the resting state, 0-back (control) task, and 2-back task. The phase lag index (PLI) between EEG channels was computed and then weighted and undirected network was constructed based on the PLI matrix. The effects of aging on network topology were examined using a brain connectivity toolbox. The results showed that age-related alteration was more prominent when the 2-back task was engaged, especially in the theta band. For the younger adults, the WM task evoked a significant increase in the clustering coefficient of the beta-band functional connectivity network, which was absent in the older adults. Furthermore, significant correlations were observed between the behavioral performance of WM and EEG metrics in the theta and gamma bands, suggesting the potential use of those measures as biomarkers for the evaluation of cognitive training, for instance. Taken together, our findings shed further light on the underlying mechanism of WM in physiological aging and suggest that different EEG frequencies appear to have distinct functional correlates in cognitive aging. Analysis of inter-regional synchronization and topological characteristics based on graph theory is thus an appropriate way to explore natural age-related changes in the human brain.

Complexity of Wake Electroencephalography Correlates With Slow Wave Activity After Sleep Onset.

Sleep electroencephalography (EEG) provides an opportunity to study sleep scientifically, whose chaotic, dynamic, complex, and dissipative nature implies that non-linear approaches could uncover some mechanism of sleep. Based on well-established complexity theories, one hypothesis in sleep medicine is that lower complexity of brain waves at pre-sleep state can facilitate sleep initiation and further improve sleep quality. However, this has never been studied with solid data. In this study, EEG collected from healthy subjects was used to investigate the association between pre-sleep EEG complexity and sleep quality. Multiscale entropy analysis (MSE) was applied to pre-sleep EEG signals recorded immediately after light-off (while subjects were awake) for measuring the complexities of brain dynamics by a proposed index, CI1-30. Slow wave activity (SWA) in sleep, which is commonly used as an indicator of sleep depth or sleep intensity, was quantified based on two methods, traditional Fast Fourier transform (FFT) and ensemble empirical mode decomposition (EEMD). The associations between wake EEG complexity, sleep latency, and SWA in sleep were evaluated. Our results demonstrated that lower complexity before sleep onset is associated with decreased sleep latency, indicating a potential facilitating role of reduced pre-sleep complexity in the wake-sleep transition. In addition, the proposed EEMD-based method revealed an association between wake complexity and quantified SWA in the beginning of sleep (90 min after sleep onset). Complexity metric could thus be considered as a potential indicator for sleep interventions, and further studies are encouraged to examine the application of EEG complexity before sleep onset in populations with difficulty in sleep initiation. Further studies may also examine the mechanisms of the causal relationships between pre-sleep brain complexity and SWA, or conduct comparisons between normal and pathological conditions.

Development and validation of a highly sensitive LC-MS/MS method for determination of brain active agent dianhydrogalactitol in mouse plasma and tissues: Application to a pharmacokinetic study.

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitative analysis of 1,2:5,6-dianhydrogalactitol (DAG) in mouse plasma and tissues. Sodium diethyldithiocarbamate (DDTC) was used as the derivatization reagent to improve its LC-MS/MS behavior. Analytes were separated on a Welch Ultimate XB-CN column with a mobile phase consisting of acetonitrile and 0.1% formic acid solution (65:35). The MS analysis was conducted by positive electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity (r2 > 0.9958) was observed over the concentration range of 1-1000 ng/mL in plasma and tissue homogenates (brain, liver, heart, spleen, lung and kidney). The intra- and inter-batch precision and accuracy of DAG in plasma and brain samples were all within the acceptable limits. The extraction recovery was stable and no significant matrix effects were observed. The method was successfully applied to study the pharmacokinetic and tissue distribution of DAG in mice after intravenous administration. DAG could cross the blood-brain barrier and had limited liver distribution. Rat primary hepatocytes in vitro experiments demonstrated that DAG had a safe profile in liver.