Dual stimuli-responsive and sustained drug delivery NanoSensoGel formulation for prevention of cisplatin-induced ototoxicity.

Cisplatin (CisPt)-induced ototoxicity (CIO) is delineated as a consequence of CisPt-induced intracellular generation of reactive oxygen species (ROS) which can be circumvented by Bucillamine (BUC; an antioxidant drug with sulfhydryl groups) and Diltiazem (DLT, L-type calcium channel blocker). However, its effective accumulation in the Organ of Corti and cell cytoplasm is desired. Therefore, a biocompatible BUC- and DLT-nanoparticles (NPs)-impregnated dual stimuli-responsive formulation (NanoSensoGel) presented here with ROS- and thermo-responsive properties for the sustained and receptive delivery of drugs. The ROS-responsive polypropylene sulfide- methyl polyethylene glycol-2000 (PPS-mPEG2000) polymer was rationally designed, synthesized, and characterized to fabricate BUC- and DLT-loaded PPS-mPEG2000-NPs (BUC- and DLT-NPs). The fabricated BUC- and DLT-NPs showed efficient cellular uptake, intracellular delivery, ROS responsiveness, and cytoprotective effect which was characterized using cellular internalization, intracellular ROS, mitochondrial superoxide, and Caspase 3/7 assays on the House Ear Institute-Organ of Corti-1 (HEI-OC1) cells. The composite NanoSensoGel (i.e., ROS-responsive BUC- and DLT-NPs suspended in the thermo-responsive hydrogel) present in a sol state at room temperature and turned to gel above 33°C, which could be essential for retaining the formulation at the target site for long-term release. The NanoSensoGel showed sustained release of BUC and DLT following Fickian release diffusion kinetics. Overall, a novel NanoSensoGel formulation developed in this study has demonstrated its great potential in delivering therapeutics in the inner ear for prophylactic treatment of CIO, and associated hearing loss.

Role of ERK1/2 Signaling in Cinnabarinic Acid-Driven Stanniocalcin 2-Mediated Protection against Alcohol-Induced Apoptosis.

We have previously shown that a bona fide aryl hydrocarbon receptor (AhR) agonist, cinnabarinic acid (CA), protects against alcohol-induced hepatocyte apoptosis via activation of a novel AhR target gene, stanniocalcin 2 (Stc2). Stc2 translates to a secreted disulfide-linked hormone, STC2, known to function in cell development, calcium and phosphate regulation, angiogenesis, and antiapoptosis-albeit the comprehensive mechanism by which the CA-AhR-STC2 axis confers antiapoptosis is yet to be characterized. In this study, using RNA interference library screening, downstream antiapoptotic molecular signaling components involved in CA-induced STC2-mediated protection against ethanol-induced apoptosis were investigated. RNA interference library screening of kinases and phosphatases in Hepa1 cells and subsequent pathway analysis identified mitogen-activated protein kinase (MAPK) signaling as a critical molecular pathway involved in CA-mediated protection. Specifically, phosphorylation of ERK1/2 was induced in response to CA treatment without alterations in p38 and JNK signaling pathways. Silencing Stc2 in Hepa1 cells and in vivo experiments performed in Stc2-/- (Stc2 knockout) mice, which failed to confer CA-mediated protection against ethanol-induced apoptosis, showed abrogation of ERK1/2 activation, underlining the significance of ERK1/2 signaling in CA-STC2-mediated protection. In conclusion, activation of ERK1/2 signaling in CA-driven AhR-dependent Stc2-mediated protection represents a novel mechanism of protection against acute alcohol-induced apoptosis. SIGNIFICANCE STATEMENT: Previous studies have shown the role of stanniocalcin 2 (Stc2) in cinnabarinic acid (CA)-mediated protection against alcohol-induced apoptosis. Here, using RNA interference library screening and subsequent in vivo studies, the functional significance of ERK1/2 activation in CA-induced Stc2-mediated protection against acute ethanol-induced apoptosis was identified. This study is thus significant as it illustrates a comprehensive downstream mechanism by which CA-induced Stc2 protects against alcoholic liver disease.

The Use of the QbD Approach to Optimize the Co-Loading of Simvastatin and Doxorubicin in Liposomes for a Synergistic Anticancer Effect.

The present study aimed to optimize a liposomal formulation co-encapsulating simvastatin (SIM) and doxorubicin (DOX) that has future perspectives in anticancer therapy. The optimization process was performed by implementing the Quality by Design concept and by considering the results of a previous screening study. Failure Mode and Effects Analysis was used for the identification of the potential critical factors, i.e., phospholipid, SIM and DOX concentration, which were assessed in an optimization experimental design with the purpose of designing an optimal formulation. The optimal formulation, meeting the established quality profile, was additionally characterized in terms of the release profile and antiproliferative effects. During dissolution studies, a novel chronoamperometric method was used for the simultaneous quantification of SIM and DOX. The obtained data confirmed the similarity of this method with a validated HPLC method. The anticancer potential of the optimal formulation was tested against two human cancerous cell lines, namely T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line. The results highlighted that the antiproliferative effect of the optimal formulation is concentration dependent and favors a synergistic effect of the two drugs.

Cinnabarinic Acid Provides Hepatoprotection Against Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic condition in which excess lipids accumulate in the liver and can lead to a range of progressive liver disorders including non-alcoholic steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. While lifestyle and diet modifications have proven to be effective as NAFLD treatments, they are not sustainable in the long-term, and currently no pharmacological therapies are approved to treat NAFLD. Our previous studies demonstrated that cinnabarinic acid (CA), a novel endogenous Aryl hydrocarbon Receptor (AhR) agonist, activates the AhR target gene, Stanniocalcin 2, and confers cytoprotection against a plethora of ER/oxidative stressors. In this study, the hepatoprotective and anti-steatotic properties of CA were examined against free fatty-acid-induced in vitro and high-fat-diet fed in vivo NAFLD models. The results demonstrated that CA treatment significantly lowered weight gain and attenuated hepatic lipotoxicity both before and after the established fatty liver, thereby protecting against steatosis, inflammation, and liver injury. CA mitigated intracellular free fatty acid uptake concomitant with the downregulation of CD36/fatty acid translocase. Genes involved in fatty acid and triglyceride synthesis were also downregulated in response to CA treatment. Additionally, suppressing AhR and Stc2 expression using RNA interference in vitro verified that the hepatoprotective effects of CA were absolutely dependent on both AhR and its target, Stc2. Collectively, our results demonstrate that the endogenous AhR agonist, CA, confers hepatoprotection against NAFLD by regulating hepatic fatty acid uptake and lipogenesis. SIGNIFICANCE STATEMENT: In this study using in vitro and in vivo models, we demonstrate that cinnabarinic acid (CA), an endogenous AhR agonist, provides protection against non-alcoholic fatty liver disease. CA bestows cytoprotection against steatosis and liver injury by controlling expression of several key genes associated with lipid metabolism pathways, limiting the hepatic lipid uptake, and controlling liver inflammation. Moreover, CA-induced hepatoprotection is absolutely dependent on AhR and Stc2 expression.

Decoding Cinnabarinic Acid-Specific Stanniocalcin 2 Induction by Aryl Hydrocarbon Receptor.

Aryl hydrocarbon receptor (AhR) is a ligand-mediated transcription factor known for regulating response to xenobiotics, including prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the activation of CYP1A1 expression. Upon ligand-binding, AhR translocates to the nucleus, interacts with the AhR nuclear translocator, and binds to xenobiotic response elements (XREs; GCGTG) present in the promoter region of AhR-regulated genes. Recently, we identified a novel tryptophan catabolite, cinnabarinic acid (CA), as an endogenous AhR agonist capable of activating expression of AhR target gene stanniocalcin 2 (stc2). The CA-driven stc2 induction bestowed cytoprotection against hepatotoxicity in an AhR-dependent manner. Interestingly, only CA but not TCDD was able to induce stc2 expression in liver, and CA was unable to upregulate the TCDD responsive cyp1a1 gene. In this report, we identified CA-specific histone H4 lysine 5 acetylation and H3 lysine 79 methylation at the AhR-bound stc2 promoter. Moreover, histone H4 lysine 5 acetylation writer, activating transcription factor 2 (Atf2), and H3 lysine 79 methylation writer, disruptor of telomeric silencing 1-like histone lysine methyltransferase (Dot1l), were interacting with the AhR complex at the stc2 promoter exclusively in response to CA treatment concurrent with the histone epigenetic marks. Suppressing Atf2 and Dot1l expression using RNA interference confirmed their role in stc2 expression. CRISPR/Cas9-assisted replacement of cyp1a1 promoter-encompassing XREs with stc2 promoter XREs resulted in CA-dependent induction of cyp1a1, underlining a fundamental role of quaternary structure of XRE sequence in agonist-specific gene regulation. In conclusion, CA-driven recruitment of specific chromatin regulators to the AhR complex and resulting histone epigenetic modifications may serve as a molecular basis for agonist-specific stc2 regulation by AhR. SIGNIFICANCE STATEMENT: Results reported here provide a mechanistic explanation for the agonist-specific differential gene regulation by identifying interaction of aryl hydrogen receptor with specific chromatin regulators concomitant with unique histone epigenetic marks. This study also demonstrated that the agonist-specific target-gene expression can be transferred with the gene-specific promoter xenobiotic response element-sequence in the context of chromatin architecture.

Gemcitabine Direct Electrochemical Detection from Pharmaceutical Formulations Using a Boron-Doped Diamond Electrode.

The development of fast and easy-to-use methods for gemcitabine detection is of great interest for pharmaceutical formulation control in both research laboratories and hospitals. In this study, we report a simple, fast and direct electrochemical method for gemcitabine detection using a boron-doped diamond electrode. The electrochemical oxidation of gemcitabine on a boron-doped diamond electrode was found to be irreversible in differential pulse voltammetry, and scan rate influence studies demonstrated that the process is diffusion-controlled. The influence of the pH and supporting electrolytes were also tested, and the optimized differential pulse voltammetry method was linear in the range of 2.5-50 µg/mL, with a detection limit of 0.85 µg/mL in phosphate-buffered saline (pH 7.4; 0.1 M). An amperometric method was also optimized for gemcitabine detection. The linear range of the method was 0.5-65 µg/mL in phosphate-buffered saline of pH 7.4 as well as pH 5.5, the limit of detection being 0.15 µg/mL. The optimized differential pulse voltammetry and amperometric detection strategies were successfully applied to pharmaceutical formulations, and the results were compared to those obtained by high-performance liquid chromatography and UV-Vis spectrophotometry with good correlations.

An Electrochemical Strategy for the Simultaneous Detection of Doxorubicin and Simvastatin for Their Potential Use in the Treatment of Cancer.

The aim of this study was to develop a disposable, simple, fast, and sensitive sensor for the simultaneous electrochemical detection of doxorubicin (DOX) and simvastatin (SMV), which could be used in preclinical studies for the development of new pharmaceutical formulations for drug delivery. Firstly, the electrochemical behavior of each molecule was analyzed regarding the influence of electrode material, electrolyte solution, and scan rate. After this, the proper electrode material, electrolyte solution, and scan rate for both active substances were chosen, and a linear sweep voltammetry procedure was optimized for simultaneous detection. Two chronoamperometry procedures were tested, one for the detection of DOX in the presence of SMV, and the other one for the detection of DOX and SMV together. Finally, calibration curves for DOX and SMV in the presence of each other were obtained using both electrochemical methods and the results were compared. The use of amperometry allowed for a better limit of detection (DOX: 0.1 µg/mL; SMV: 0.7 µg/mL) than the one obtained in voltammetry (1.5 µg/mL for both drugs). The limits of quantification using amperometry were 0.5 µg/mL for DOX (dynamic range: 0.5-65 µg/mL) and 2 µg/mL for SMV (dynamic range: 2-65 µg/mL), while using voltammetry 1 µg/mL was obtained for DOX (dynamic range: 1-100 µg/mL) and 5 µg/mL for SMV (dynamic range: 5-100 µg/mL). This detection strategy represents a promising tool for the analysis of new pharmaceutical formulations for targeted drug delivery containing both drugs, whose association was proven to bring benefits in the treatment of cancer.