Journal of Pharmaceutical Analysis

Automated closed-cell production platform solves dilemma of industrial-scale manufacturing for mesenchymal stromal cell-based therapy

Yirui Feng, Bin Wang

2023, 13(11): 1233-1234. doi: 10.1016/j.jpha.2023.08.017

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Stage-specific treatment of colorectal cancer: A microRNA-nanocomposite approach

Adewale Oluwaseun Fadaka, Taiwo Akinsoji, Ashwil Klein, Abram Madimabe Madiehe, Mervin Meyer, Marshall Keyster, Lucky Mashudu Sikhwivhilu, Nicole Remaliah Samantha Sibuyi

2023, 13(11): 1235-1251. doi: 10.1016/j.jpha.2023.07.008

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Colorectal cancer (CRC) is among the leading causes of cancer mortality. The lifetime risk of developing CRC is about 5% in adult males and females. CRC is usually diagnosed at an advanced stage, and at this point therapy has a limited impact on cure rates and long-term survival. Novel and/or improved CRC therapeutic options are needed. The involvement of microRNAs (miRNAs) in cancer development has been reported, and their regulation in many oncogenic pathways suggests their potent tumor suppressor action. Although miRNAs provide a promising therapeutic approach for cancer, challenges such as biodegradation, specificity, stability and toxicity, impede their progression into clinical trials. Nanotechnology strategies offer diverse advantages for the use of miRNAs for CRC-targeted delivery and therapy. The merits of using nanocarriers for targeted delivery of miRNA-formulations are presented herein to highlight the role they can play in miRNA-based CRC therapy by targeting different stages of the disease.

Biosensors for waterborne virus detection: Challenges and strategies

Xixi Song, Zina Fredj, Yuqiao Zheng, Hongyong Zhang, Guoguang Rong, Sumin Bian, Mohamad Sawan

2023, 13(11): 1252-1268. doi: 10.1016/j.jpha.2023.08.020

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Waterborne viruses that can be harmful to human health pose significant challenges globally, affecting health care systems and the economy. Identifying these waterborne pathogens is essential for preventing diseases and protecting public health. However, handling complex samples such as human and wastewater can be challenging due to their dynamic and complex composition and the ultralow concentration of target analytes. This review presents a comprehensive overview of the latest breakthroughs in waterborne virus biosensors. It begins by highlighting several promising strategies that enhance the sensing performance of optical and electrochemical biosensors in human samples. These strategies include optimizing bioreceptor selection, transduction elements, signal amplification, and integrated sensing systems. Furthermore, the insights gained from biosensing waterborne viruses in human samples are applied to improve biosensing in wastewater, with a particular focus on sampling and sample pretreatment due to the dispersion characteristics of waterborne viruses in wastewater. This review suggests that implementing a comprehensive system that integrates the entire waterborne virus detection process with high-accuracy analysis could enhance virus monitoring. These findings provide valuable insights for improving the effectiveness of waterborne virus detection, which could have significant implications for public health and environmental management.

Development status of novel spectral imaging techniques and application to traditional Chinese medicine

Qi Wang, Yong Zhang, Baofeng Yang

2023, 13(11): 1269-1280. doi: 10.1016/j.jpha.2023.07.007

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Traditional Chinese medicine (TCM) is a treasure of the Chinese nation, providing effective solutions to current medical requisites. Various spectral techniques are undergoing continuous development and provide new and reliable means for evaluating the efficacy and quality of TCM. Because spectral techniques are noninvasive, convenient, and sensitive, they have been widely applied to in vitro and in vivo TCM evaluation systems. In this paper, previous achievements and current progress in the research on spectral technologies (including fluorescence spectroscopy, photoacoustic imaging, infrared thermal imaging, laser-induced breakdown spectroscopy, hyperspectral imaging, and surface enhanced Raman spectroscopy) are discussed. The advantages and disadvantages of each technology are also presented. Moreover, the future applications of spectral imaging to identify the origins, components, and pesticide residues of TCM in vitro are elucidated. Subsequently, the evaluation of the efficacy of TCM in vivo is presented. Identifying future applications of spectral imaging is anticipated to promote medical research as well as scientific and technological explorations.

Oridonin restores hepatic lipid homeostasis in an LXRα-ATGL/EPT1 axis-dependent manner

Yulian Chen, Huanguo Jiang, Zhikun Zhan, Jindi Lu, Tanwei Gu, Ping Yu, Weimin Liang, Xi Zhang, Shilong Zhong, Lan Tang

2023, 13(11): 1281-1295. doi: 10.1016/j.jpha.2023.08.010

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Hepatosteatosis is characterized by abnormal accumulation of triglycerides (TG), leading to prolonged and chronic inflammatory infiltration. To date, there is still a lack of effective and economical therapies for hepatosteatosis. Oridonin (ORI) is a major bioactive component extracted from the traditional Chinese medicinal herb Rabdosia rubescens. In this paper, we showed that ORI exerted significant protective effects against hepatic steatosis, inflammation and fibrosis, which was dependent on LXRα signaling. It is reported that LXRα regulated lipid homeostasis between triglyceride (TG) and phosphatidylethanolamine (PE) by promoting ATGL and EPT1 expression. Therefore, we implemented the lipidomic strategy and luciferase reporter assay to verify that ORI contributed to the homeostasis of lipids via the regulation of the ATGL gene associated with TG hydrolysis and the EPT1 gene related to PE synthesis in a LXRα-dependent manner, and the results showed the TG reduction and PE elevation. In detail, hepatic TG overload and lipotoxicity were reversed after ORI treatment by modulating the ATGL and EPT1 genes, respectively. Taken together, the data provide mechanistic insights to explain the bioactivity of ORI in attenuating TG accumulation and cytotoxicity and introduce exciting opportunities for developing novel natural activators of the LXRα-ATGL/EPT1 axis for pharmacologically treating hepatosteatosis and metabolic disorders.

Ginsenoside Rg5 enhances the radiosensitivity of lung adenocarcinoma via reducing HSP90-CDC37 interaction and promoting client protein degradation

Hansong Bai, Jiahua Lyu, Xinyu Nie, Hao Kuang, Long Liang, Hongyuan Jia, Shijie Zhou, Churong Li, Tao Li

2023, 13(11): 1296-1308. doi: 10.1016/j.jpha.2023.06.004

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Ginsenoside Rg5 is a rare ginsenoside showing promising tumor-suppressive effects. This study aimed to explore its radio-sensitizing effects and the underlying mechanisms. Human lung adenocarcinoma cell lines A549 and Calu-3 were used for in vitro and in vivo analysis. Bioinformatic molecular docking prediction and following validation by surface plasmon resonance (SPR) technology, cellular thermal shift assay (CETSA), and isothermal titration calorimetry (ITC) were conducted to explore the binding between ginsenoside Rg5 and 90 kD heat shock protein alpha (HSP90α). The effects of ginsenoside Rg5 on HSP90-cell division cycle 37 (CDC37) interaction, the client protein stability, and the downstream regulations were further explored. Results showed that ginsenoside Rg5 could induce cell-cycle arrest at the G1 phase and enhance irradiation-induced cell apoptosis. It could bind to HSP90α with a high affinity, but the affinity was drastically decreased by HSP90α Y61A mutation. Co-immunoprecipitation (Co-IP) and ITC assays confirmed that ginsenoside Rg5 disrupts the HSP90-CDC37 interaction in a dose-dependent manner. It reduced irradiation-induced upregulation of the HSP90-CDC37 client proteins, including SRC, CDK4, RAF1, and ULK1 in A549 cell-derived xenograft (CDX) tumors. Ginsenoside Rg5 or MRT67307 (an IKKε/TBK1 inhibitor) pretreatment suppressed irradiation-induced elevation of the LC3-II/β ratio and restored irradiation-induced downregulation of p62 expression. In A549 CDX tumors, ginsenoside Rg5 treatment suppressed LC3 expression and enhanced irradiation-induced DNA damage. In conclusion, ginsenoside Rg5 may be a potential radiosensitizer for lung adenocarcinoma. It interacts with HSP90α and reduces the binding between HSP90 and CDC37, thereby increasing the ubiquitin-mediated proteasomal degradation of the HSP90-CDC37 client proteins.

Canonical transient receptor potential channel 1 aggravates myocardial ischemia-and-reperfusion injury by upregulating reactive oxygen species

Hui-Nan Zhang, Meng Zhang, Wen Tian, Wei Quan, Fan Song, Shao-Yuan Liu, Xiao-Xiao Liu, Dan Mo, Yang Sun, Yuan-Yuan Gao, Wen Ye, Ying-Da Feng, Chang-Yang Xing, Chen Ye, Lei Zhou, Jing-Ru Meng, Wei Cao, Xiao-Qiang Li

2023, 13(11): 1309-1325. doi: 10.1016/j.jpha.2023.08.018

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The canonical transient receptor potential channel (TRPC) proteins form Ca²⁺-permeable cation channels that are involved in various heart diseases. However, the roles of specific TRPC proteins in myocardial ischemia/reperfusion (I/R) injury remain poorly understood. We observed that TRPC1 and TRPC6 were highly expressed in the area at risk (AAR) in a coronary artery ligation induced I/R model. Trpc1^-/- mice exhibited improved cardiac function, lower serum Troponin T and serum creatine kinase level, smaller infarct volume, less fibrotic scars, and fewer apoptotic cells after myocardial-I/R than wild-type or Trpc6^-/- mice. Cardiomyocyte-specific knockdown of Trpc1 using adeno-associated virus 9 mitigated myocardial I/R injury. Furthermore, Trpc1 deficiency protected adult mouse ventricular myocytes (AMVMs) and HL-1 cells from death during hypoxia/reoxygenation (H/R) injury. RNA-sequencing-based transcriptome analysis revealed differential expression of genes related to reactive oxygen species (ROS) generation in Trpc1^-/- cardiomyocytes. Among these genes, oxoglutarate dehydrogenase-like (Ogdhl) was markedly downregulated. Moreover, Trpc1 deficiency impaired the calcineurin (CaN)/nuclear factor-kappa B (NF-κB) signaling pathway in AMVMs. Suppression of this pathway inhibited Ogdhl upregulation and ROS generation in HL-1 cells under H/R conditions. Chromatin immunoprecipitation assays confirmed NF-κB binding to the Ogdhl promoter. The cardioprotective effect of Trpc1 deficiency was canceled out by overexpression of NF-κB and Ogdhl in cardiomyocytes. In conclusion, our findings reveal that TRPC1 is upregulated in the AAR following myocardial I/R, leading to increased Ca²⁺ influx into associated cardiomyocytes. Subsequently, this upregulates Ogdhl expression through the CaN/NF-κB signaling pathway, ultimately exacerbating ROS production and aggravating myocardial I/R injury.

Nanoscale coordination polymer Fe-DMY downregulating Poldip2-Nox4-H₂O₂ pathway and alleviating diabetic retinopathy

Si-Yu Gui, Xin-Chen Wang, Zhi-Hao Huang, Mei-Mei Li, Jia-Hao Wang, Si-Yin Gui, Gan-Hua Zhang, Yao Lu, Li-Ming Tao, Hai-Sheng Qian, Zheng-Xuan Jiang

2023, 13(11): 1326-1345. doi: 10.1016/j.jpha.2023.05.002

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Diabetic retinopathy (DR) is a prevalent microvascular complication of diabetes and the leading cause of blindness and severe visual impairment in adults. The high levels of glucose trigger multiple intracellular oxidative stress pathways, such as POLDIP2, resulting in excessive reactive oxygen species (ROS) production and increased expression of vascular cell adhesion molecule-1 (VCAM-1), hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF), causing microvascular dysfunction. Dihydromyricetin (DMY) is a natural flavonoid small molecule antioxidant. However, it exhibits poor solubility in physiological environments, has a short half-life in vivo, and has low oral bioavailability. In this study, we present, for the first time, the synthesis of ultra-small Fe-DMY nano-coordinated polymer particles (Fe-DMY NCPs), formed by combining DMY with low-toxicity iron ions. In vitro and in vivo experiments confirm that Fe-DMY NCPs alleviate oxidative stress-induced damage to vascular endothelial cells by high glucose, scavenge excess ROS, and improve pathological features of DR, such as retinal vascular leakage and neovascularization. Mechanistic validation indicates that Fe-DMY NCPs can inhibit the activation of the Poldip2-Nox4-H₂O₂ signaling pathway and downregulate vital vascular function indicators such as VCAM-1, HIF-1α, and VEGF. These findings suggest that Fe-DMY NCPs could serve as a safe and effective antioxidant and microangio-protective agent, with the potential as a novel multimeric drug for DR therapy.

Nylon 6-cellulose composite hosted in a hypodermic needle: Biofluid extraction and analysis by ambient mass spectrometry in a single device

Jaime Millán-Santiago, Rafael Lucena, Soledad Cárdenas

2023, 13(11): 1346-1352. doi: 10.1016/j.jpha.2023.06.015

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This study proposes a hypodermic needle (HN) as a sorbent holder and an electrospray (ESI) emitter, thus combining extraction and analysis in a single device. A novel nylon 6-cellulose (N6-Cel) composite sorbent is proposed to extract methadone from oral fluid samples. The cellulosic substrate provides the composite with high porosity, permitting the flow-through of the sample, while the polyamide contributes to the extraction of the analyte. The low price of the devices (considering the holder and the sorbent) contributes to the affordability of the method, and their small size allows easy transportation, opening the door to on-site extractions. Under the optimum conditions, the analyte can be determined by high-resolution ambient ionization mass spectrometry at a limit of detection (LOD) as low as 0.3 μg/L and precision (expressed as relative standard deviation, RSD) better than 9.3%. The trueness, expressed as relative recovery (RR), ranged from 90% to 109%. As high-resolution mass spectrometers are not available in many laboratories, the method was also adapted to low-resolution spectrometers. In this sense, the direct infusion of the eluates in a triple quadrupole-mass spectrometry provided an LOD of 2.2 μg/L. The RSD was better than 5.3%, and the RR ranged from 96% to 121%.

Polarity-regulated derivatization-assisted LC-MS method for amino-containing metabolites profiling in gastric cancer

Jie Han, Shilin Gong, Xiqing Bian, Yun Qian, Guilan Wang, Na Li, Jian-Lin Wu

2023, 13(11): 1353-1364. doi: 10.1016/j.jpha.2023.06.009

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Amino-containing compounds, including amino acids, aliphatic amines, aromatic amines, small peptides and catecholamines, are involved in various biological processes and play vital roles in multiple metabolic pathways. Previous studies indicated that some amino-containing metabolites are significant diagnostic and prognostic biomarkers of gastric cancer. However, the discovery of precise biomarkers for the preoperative diagnosis of gastric cancer is still in an urgent need. Herein, we established a polarity-regulated derivatization method coupled with liquid chromatography-mass spectrometry (LC-MS) for amino-containing metabolites profiling in the serum samples of patients with gastric cancer and healthy controls, based on our newly designed and synthesized derivatization reagent (S)-3-(1-(diisopropoxyphosphoryl) pyrrolidine-2-carboxamido)-N-hydroxysuccinimidyl ester (3-DP-NHS). Enhanced separation efficiency and detection sensitivity for amino-containing metabolites were achieved after derivatization. This method exhibited good linearity, recovery, intra- and inter-day precision and accuracy. Only 5 μL serum is needed for untargeted analysis, enabling 202 amino-containing metabolites to be detected. Statistical analysis revealed altered amino acid metabolisms in patients with gastric cancer. Furthermore, ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) analysis quantification revealed increased serum levels of tryptamine and decreased concentrations of arginine and tryptophan in patients with gastric cancer. Receiver operating characteristic (ROC) curves indicated that an increased tryptamine/tryptophan ratio could serve as a potential biomarker for gastric cancer diagnosis. This study demostrated the possibility of using serum amino acid biomarkers for gastric cancer diagnosis, providing new avenues for the treatment of gastric cancer.

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

Li Liu, Yu-Han Lu, Min-Dan Wang, Qun-Fei Zhao, Xiu-Ping Chen, Hang Yin, Chen-Guo Feng, Fang Zhang

2023, 13(11): 1365-1373. doi: 10.1016/j.jpha.2023.08.016

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In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R² = 0.9981-0.9999), precision (interday precision of 1.6%–19.0% and intraday precision of 1.4%–19.8%) and accuracy (83.4%–115.7%). Moreover, the recovery assessments in tissues (82.5%–107.3%) and in cells (98.1%–118.9%) with GSH-¹³C2, ¹⁵N, and Cys-¹⁵N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

2023 Vol. 13, No. 11