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Psoriasis, a chronic immune-mediated skin disorder affecting over 125 million people globally, is characterized by abnormal keratinocyte proliferation and immune cell infiltration. Photodynamic therapy (PDT) remains underutilized in the treatment of psoriasis despite its potential as a promising and effective therapeutic approach. This study aimed to explore the efficacy of zinc phthalocyanine (ZnPc) and its sugar conjugates as potential antipsoriatic agents. We successfully synthesized protected and unprotected sugar-conjugated zinc phthalocyanines and evaluated their potential against cytokine-stimulated HaCaT keratinocytes, as well as an established IMQ psoriasis-like in vivo model. Tetrasubstituted protected glucose–ZnPc (Glu-4-ZnPc-P) demonstrated superior phototoxicity (IC50 = 2.55 µM) compared to unprotected glucose conjugate (IC50 = 22.7 µM), protected galactose–ZnPc (IC50 = 7.13 µM), and free ZnPc in cytokine-stimulated HaCaT cells (IC50 = 5.84 µM). Cellular uptake analysis revealed that IL-17A, a cytokine that plays a central role in the pathogenesis of psoriasis, enhanced unprotected Glu-4-ZnPc uptake by 56.3%, while GLUT1 inhibitor BAY-876 reduced its accumulation by 23.8%. Intracellular ROS generation following Glu-4-ZnPc-P-PDT was significantly increased after stimulation with IL-17A, correlating with in vitro photocytotoxicity. In vivo PDT using Glu-4-ZnPc-P exhibited significant improvement in Psoriasis Area and Severity Index (PASI), inhibiting splenomegaly and restoring normal skin morphology. This study highlights sugar-conjugated zinc phthalocyanines as potential candidates for targeted PDT in psoriasis, providing a basis for further clinical investigations. [ABSTRACT FROM AUTHOR]

Background: Pyrazinamide is a Biopharmaceutical Classification System class III antibiotic indicated for active tuberculosis. Methods: In the present work, pyrazinamide-loaded biodegradable polymeric nanoparticles (PNPs) based dry powder inhaler were developed using the double emulsion solvent evaporation technique and optimized using design of experiments to provide direct pulmonary administration with minimal side effects. Batches were characterized for various physicochemical and aerosol performance properties. Results: Optimized batch exhibited particle size of 284.5 nm, % entrapment efficiency of 71.82%, polydispersibility index of 0.487, zeta potential of −17.23 mV, and in vitro drug release at 4 hours of 79.01%. Spray-dried PNPs were evaluated for drug content, in vitro drug release, and kinetics. The particle mass median aerodynamic diameter was within the alveolar region's range (2.910 μm). In the trachea and lung, there was a 2.5- and 1.2-fold increase in in vivo deposition with respect to pure drug deposition, respectively. In vitro drug uptake findings showed that alveolar macrophages with pyrazinamide PNPs had a considerably higher drug concentration. Furthermore, accelerated stability studies were carried out for the optimized batch. Results indicated no significant change in the evaluation parameters, which showed stability of the formulation for at least a 6-month period. Conclusion: PNPs prepared using biodegradable polymers exhibited efficient pulmonary drug delivery with decent stability. [ABSTRACT FROM AUTHOR]

Psoriasis, a chronic immune-mediated skin disorder affecting over 125 million people globally, is characterized by abnormal keratinocyte proliferation and immune cell infiltration. Photodynamic therapy (PDT) remains underutilized in the treatment of psoriasis despite its potential as a promising and effective therapeutic approach. This study aimed to explore the efficacy of zinc phthalocyanine (ZnPc) and its sugar conjugates as potential antipsoriatic agents. We successfully synthesized protected and unprotected sugar-conjugated zinc phthalocyanines and evaluated their potential against cytokine-stimulated HaCaT keratinocytes, as well as an established IMQ psoriasis-like in vivo model. Tetrasubstituted protected glucose–ZnPc (Glu-4-ZnPc-P) demonstrated superior phototoxicity (IC50 = 2.55 µM) compared to unprotected glucose conjugate (IC50 = 22.7 µM), protected galactose–ZnPc (IC50 = 7.13 µM), and free ZnPc in cytokine-stimulated HaCaT cells (IC50 = 5.84 µM). Cellular uptake analysis revealed that IL-17A, a cytokine that plays a central role in the pathogenesis of psoriasis, enhanced unprotected Glu-4-ZnPc uptake by 56.3%, while GLUT1 inhibitor BAY-876 reduced its accumulation by 23.8%. Intracellular ROS generation following Glu-4-ZnPc-P-PDT was significantly increased after stimulation with IL-17A, correlating with in vitro photocytotoxicity. In vivo PDT using Glu-4-ZnPc-P exhibited significant improvement in Psoriasis Area and Severity Index (PASI), inhibiting splenomegaly and restoring normal skin morphology. This study highlights sugar-conjugated zinc phthalocyanines as potential candidates for targeted PDT in psoriasis, providing a basis for further clinical investigations.

The present investigations aimed to compare the efficiency of PAMAM G4 (PG4) and PEGylated PAMAM G4 (PPG4) dendrimers as novel nanocarriers for the treatment of HIV-1. Synthesized PG4 and PPG4 dendrimers were confirmed by electrospray ionization and particle size with its morphology. The anti-human immunodeficiency virus (HIV) drug efavirenz (EFV) with a booster dose of ritonavir (RTV) was encapsulated into PG4 and PPG4 formerly noted as PG4ER and PPG4ER, respectively. Further, evaluated for dendrimers mediated solubilization, drug release, cytotoxicity, drug uptake, plasma, and tissue pharmacokinetics, and histopathology. PG4ER and PPG4ER both promoted a prolonged release of EFV in weakly acidic pH 4 up to 84 h and 132 h, respectively. The results of the cytotoxicity assay and drug uptake study showed that PPG4ER was safe and biocompatible up to 12.5 µg/ml. The plasma pharmacokinetic profile of EFV and RTV was significantly increased by PPG4ER with prolonged t1/2 up to three times as compared to free EFV–RTV and PG4ER. Histopathological analysis showed remarkably lower tissue toxicity in PPG4ER as compared to free EFV–RTV. Therefore, overall data suggested that PPG4 has a great potential for prolonged release of EFV and RTV with enhanced bioavailability and lower toxicity. [ABSTRACT FROM AUTHOR]

The plasma membrane is a lipid bilayer that establishes the outer boundary of a living cell. The composition of the lipid bilayer influences the membrane's biophysical properties, including fluidity, thickness, permeability, phase behavior, charge, elasticity, and formation of flat sheet or curved structures. Changes in the biophysical properties of the membrane can be occasioned when new entities, such as drug molecules, are partitioned in the bilayer. Therefore, assessing drugs for their effect on the biophysical properties of the lipid bilayer of a cell membrane is critical to understanding specific and non-specific drug action. Previously, we reported a non-invasive technique for real-time characterization of cellular dielectric properties, such as membrane capacitance and cytoplasmic conductivity. In this study, we discuss the potential application of the technique in assessing the biophysical properties of the cell membrane in response to interaction with amiodarone compared to aspirin/acetylsalicylic acid and glucose. Amiodarone is a potent drug used to treat cardiac arrhythmia, but it also exerts various non-specific effects. Compared to aspirin and glucose, we measured a rapid and higher magnitude increase in membrane capacitance on cells under amiodarone treatment. Increased membrane capacitance induced by aspirin and glucose quickly returned to baseline in 15 s, while amiodarone-induced increased capacitance sustained and decreased slowly, approaching baseline or another asymptotic limit in ~2.5 h. Because amiodarone has a strong lipid partitioning property, we reason that drug partitioning alters the lipid bilayer context and subsequently reduces bilayer thickness, leading to an increase in the electrical capacitance of the cell membrane. The presented microfluidic system promises a new approach to assess drug–membrane interactions and delineate specific and non-specific actions of the drug on cells. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is the most common malignancy in women worldwide. While the main systemic treatment option is anthracycline-containing chemotherapy, chemoresistance continues to be an obstacle to patient survival. Carnitine palmitoyltransferase 1C (CPT1C) has been described as a poor-prognosis marker for several tumour types, as it favours tumour growth and hinders cells from entering senescence. At the molecular level, CPT1C has been associated with lipid metabolism regulation and important lipidome changes. Since plasma membrane (PM) rigidity has been associated with reduced drug uptake, we explored whether CPT1C expression could be involved in PM remodelling and drug chemoresistance. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) lipid analysis of PM-enriched fractions of MDA-MB-231 BC cells showed that CPT1C silencing increased PM phospholipid saturation, suggesting a rise in PM rigidity. Moreover, CPT1C silencing increased cell survival against doxorubicin (DOX) treatment in different BC cells due to reduced drug uptake. These findings, further complemented by ROC plotter analysis correlating lower CPT1C expression with a lower pathological complete response to anthracyclines in patients with more aggressive types of BC, suggest CPT1C as a novel predictive biomarker for BC chemotherapy. [ABSTRACT FROM AUTHOR]

The low water solubility of baricitinib (BCT) limits the development of new formulations for the topical delivery of the drug. The aims of this study were to assess the solubility of BCT in different solvents, including Transcutol, a biocompatible permeation enhancer that is miscible in water, to evaluate the drug uptake in human skin and porcine tissues (sclera, cornea, oral, sublingual, and vaginal), and to subsequently extract the drug from the tissues so as to determine the drug recovery using in vitro techniques. Analytical methods were developed and validated for the quantification of BCT in Transcutol using absorption and fluorescence spectroscopies, which are complementary to each other and permit the detection of the drug across a broad range of concentrations. Results show that Transcutol permits an increased drug solubility, and that BCT is able to penetrate the tissues studied. The solutions of BCT in Transcutol were stable for at least one week. Hence, Transcutol may be a suitable solvent for further development of topical formulations. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus -specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics., Competing Interests: Declaration of Interests The authors declare no competing interests.

Nanoparticle deployment in drug delivery is contingent upon controlled drug loading and a desired release profile, with simultaneous biocompatibility and cellular targeting. Iron oxide nanoparticles (IONPs), being biocompatible, are used as drug carriers. However, to prevent aggregation of bare IONPs, they are coated with stabilizing agents. We hypothesize that, zwitterionic drugs like norfloxacin (NOR, a fluoroquinolone) can manifest dual functionality – nanoparticle stabilization and antibiotic activity, eliminating the need of a separate stabilizing agent. Since these drugs have different charges, depending on the surrounding pH, drug loading enhancement could be pH dependent. Hence, upon synthesizing IONPs, they were coated with NOR, either at pH 5 (predominantly as cationic, NOR+) or at pH 10 (predominantly as anionic, NOR−). We observed that, drug loading at pH 5 exceeded that at pH 10 by 4.7–5.7 times. Furthermore, only the former (pH 5 system) exhibited a desirable slower drug release profile, compared to the free drug. NOR-coated IONPs also enable a 22 times higher drug accumulation in macrophages, compared to identical extracellular concentrations of the free drug. Thus, lowering the drug coating pH to 5 imparts multiple benefits – improved IONP stability, enhanced drug coating, higher drug uptake in macrophages at reduced toxicity and slower drug release.

• A fluorescent probe, leonurine-P, was facilely prepared from coupling FITC and the small-molecule phytodrug leonurine. • Leonurine-P can be used to visualize spatiotemporal dynamics processes such as drug uptake and distribution in C. elegans and living cells. • Leonurine-P can be further explored in standard fluorescence imaging or flow cytometric platforms to allow quantitative analyses. Caenorhabditis elegans (C. elegans) has been recognized for being a useful model organism in small-molecule drug screens and drug efficacy investigation. However, there remain bottlenecks in evaluating such processes as drug uptake and distribution due to a lack of appropriate chemical tools. This study aims to prepare fluorescence-labeled leonurine as an example to monitor drug uptake and distribution of small molecule in C. elegans and living cells. FITC-conjugated leonurine (leonurine-P) was synthesized and characterized by LC/MS, NMR, UV absorption and fluorescence intensity. Leonurine-P was used to stain C. elegans and various mammalian cell lines. Different concentrations of leonurine were tested in conjunction with a competing parent molecule to determine whether leonurine-P and leonurine shared the same biological targets. Drug distribution was analyzed by imaging. Fluorometry in microplates and flow cytometry were performed for quantitative measurements of drug uptake. The UV absorption peak of leonurine-P was 490∼495 nm and emission peak was 520 nm. Leonurine-P specifically bound to endogenous protein targets in C. elegans and mammalian cells, which was competitively blocked by leonurine. The highest enrichment levels of leonurine-P were observed around 72 h following exposure in C. elegans. Leonurine-P can be used in a variety of cells to observe drug distribution dynamics. Flow cytometry of stained cells can be facilely carried out to quantitatively detect probe signals. The strategy of fluorescein-labeled drugs reported herein allows quantification of drug enrichment and visualization of drug distribution, thus illustrates a convenient approach to study phytodrugs in pharmacological contexts. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. Methods Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. Results Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. Conclusions Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.

The plasma membrane is a lipid bilayer that establishes the outer boundary of a living cell. The composition of the lipid bilayer influences the membrane’s biophysical properties, including fluidity, thickness, permeability, phase behavior, charge, elasticity, and formation of flat sheet or curved structures. Changes in the biophysical properties of the membrane can be occasioned when new entities, such as drug molecules, are partitioned in the bilayer. Therefore, assessing drugs for their effect on the biophysical properties of the lipid bilayer of a cell membrane is critical to understanding specific and non-specific drug action. Previously, we reported a non-invasive technique for real-time characterization of cellular dielectric properties, such as membrane capacitance and cytoplasmic conductivity. In this study, we discuss the potential application of the technique in assessing the biophysical properties of the cell membrane in response to interaction with amiodarone compared to aspirin/acetylsalicylic acid and glucose. Amiodarone is a potent drug used to treat cardiac arrhythmia, but it also exerts various non-specific effects. Compared to aspirin and glucose, we measured a rapid and higher magnitude increase in membrane capacitance on cells under amiodarone treatment. Increased membrane capacitance induced by aspirin and glucose quickly returned to baseline in 15 s, while amiodarone-induced increased capacitance sustained and decreased slowly, approaching baseline or another asymptotic limit in ~2.5 h. Because amiodarone has a strong lipid partitioning property, we reason that drug partitioning alters the lipid bilayer context and subsequently reduces bilayer thickness, leading to an increase in the electrical capacitance of the cell membrane. The presented microfluidic system promises a new approach to assess drug–membrane interactions and delineate specific and non-specific actions of the drug on cells.

Breast cancer (BC) is the most common malignancy in women worldwide. While the main systemic treatment option is anthracycline-containing chemotherapy, chemoresistance continues to be an obstacle to patient survival. Carnitine palmitoyltransferase 1C (CPT1C) has been described as a poor-prognosis marker for several tumour types, as it favours tumour growth and hinders cells from entering senescence. At the molecular level, CPT1C has been associated with lipid metabolism regulation and important lipidome changes. Since plasma membrane (PM) rigidity has been associated with reduced drug uptake, we explored whether CPT1C expression could be involved in PM remodelling and drug chemoresistance. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) lipid analysis of PM-enriched fractions of MDA-MB-231 BC cells showed that CPT1C silencing increased PM phospholipid saturation, suggesting a rise in PM rigidity. Moreover, CPT1C silencing increased cell survival against doxorubicin (DOX) treatment in different BC cells due to reduced drug uptake. These findings, further complemented by ROC plotter analysis correlating lower CPT1C expression with a lower pathological complete response to anthracyclines in patients with more aggressive types of BC, suggest CPT1C as a novel predictive biomarker for BC chemotherapy.

ABSTRACT The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

Background: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). Methods: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. Findings: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. Interpretation: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Objectives/Hypothesis: Lipopolysaccharide (LPS), a key component of bacterial endotoxins, activates macrophages and triggers the release of inflammatory cytokines in mammalian tissues. Recent studies have shown that intratympanic injection of LPS simulates acute otitis media (AOM) and results in morphological and functional changes in the inner ear. Here we established an AOM mouse model with LPS to investigate the uptake of ototoxic gentamicin in the inner ear, and elucidated the underlying mechanism by focusing on cochlear inflammation as a result of AOM. Study Design: Preclinical rodent animal model. Methods: Fluorescently tagged gentamicin (GTTR) was systemically administered to mice with AOM. Iba1‐positive macrophage morphology and inner ear cytokine profile were evaluated by immunofluorescence technique and a mouse cytokine array kit, respectively. Results: We observed characteristic symptoms of AOM in the LPS‐treated ears with elevated hearing thresholds indicating a conductive hearing loss. More importantly, the LPS‐induced AOM activated cochlear inflammatory responses, manifested by macrophage infiltration, particularly in the organ of Corti and the spiral ligament, in addition to the up‐regulation of proinflammatory cytokines. Meanwhile, GTTR uptake in the stria vascularis and sensory hair cells from all the LPS‐treated ears was significantly enhanced at 24, 48, and 72‐hour post‐treatment, as the most prominent enhancement was observed in the 48‐hour group. Conclusion: In summary, this study suggests that the pathological cochlea is more susceptible to ototoxic drugs, including aminoglycosides, and justified the clinical concern of aminoglycoside ototoxicity in the AOM treatment. Laryngoscope, 131:E2573–E2582, 2021 [ABSTRACT FROM AUTHOR]

In electrochemotherapy (ECT), chemotherapeutics are first administered, followed by short 100 μs monopolar pulses. However, these pulses cause pain and muscle contractions. It is thus necessary to administer muscle relaxants, general anesthesia and synchronize pulses with the heart rhythm of the patient, which makes the treatment more complex. It was suggested in ablation with irreversible electroporation, that bursts of short high-frequency bipolar pulses could alleviate these problems. Therefore, we designed our study to verify if it is possible to use high-frequency bipolar pulses (HF-EP pulses) in electrochemotherapy.

Abstract Background Aquaporins (AQPs) are a family of integral membrane channel proteins that facilitate the transport of water and other small solutes across cell membranes. AQPs appear to play crucial roles in parasite survival and represent possible drug targets for novel intervention strategy. In this work, we investigated the tissue distribution and biological roles of an aquaporin TcAQP1 in the neglected parasitic nematode Toxocara canis. Methods Recombinant C-terminal hydrophilic domain of AQP1 of T. canis (rTcAQP1c) and polyclonal antibody against rTcAQP1c were produced to analyse the tissue expression of native TcAQP1 in adult (female and male) worms using an immunohistochemical approach. RNA interference (RNAi), quantitative real-time PCR (qRT-PCR) and nematocidal assays were performed to investigate the functional roles of TcAQP1 in the adult stage of T. canis. Results Immunofluorescence analysis showed that TcAQP1 was localised predominantly in the epithelial linings of the reproductive tract and basolateral membrane of the intestine in the adult stage (female and male) of T. canis, indicating important roles in reproduction, nutrient absorption and/or osmoregulation. Treatment with silencing RNA for 24 h resulted in a significant reduction of Tc-aqp-1 mRNA level in adult T. canis, though no phenotypical change was observed. The efficient gene knockdown compromised the nematocidal activity of albendazole in vitro, suggesting the role of TcAQP1 in drug uptake. Conclusions The findings of this study provide important information about tissue expression and functional roles of TcAQP1 protein in adult T. canis. Understanding the biological functions of this protein in other developmental stages of T. canis and related parasitic nematodes would contribute to the discovery of novel diagnostic or anthelmintic targets.

Herein, we investigated the use of multimodal Raman and infrared (IR) spectroscopic microscopy for the elucidation of drug uptake and subsequent cellular responses. Firstly, we compared different methods for the analysis of the combined data. Secondly, we evaluated whether the combined analysis provided enough benefits to justify the fusion of the data. A459 cells inoculated with doxorubicin (DOX) at different times were fixed and analysed using each technique. Raman spectroscopy provided high sensitivity to DOX and enabled an accurate estimation of the drug uptake at each time point, whereas IR provided a better insight into the resultant changes in the biochemical composition of the cell. In terms of benefits of data fusion, 2D correlation analysis allowed the study of the relationship between IR and Raman variables, whereas the joint analysis of IR and Raman enabled the correlation of the different variables to be monitored over time. In summary, the complementary nature of IR and Raman makes the combination of these vibrational techniques an appealing tool to follow drug kinetics and cellular response. [ABSTRACT FROM AUTHOR]

Drug-induced transformations in disease characteristics at the cellular and molecular level offers the opportunity to predict and evaluate the efficacy of pharmaceutical ingredients whilst enabling the optimal design of new and improved drugs with enhanced pharmacokinetics and pharmacodynamics. Machine learning is a promising in-silico tool used to simulate cells with specific disease properties and to determine their response toward drug uptake. Differences in the properties of normal and infected cells, including biophysical, biochemical and physiological characteristics, plays a key role in developing fundamental cellular probing platforms for machine learning applications. Cellular features can be extracted periodically from both the drug treated, infected, and normal cells via image segmentations in order to probe dynamic differences in cell behavior. Cellular segmentation can be evaluated to reflect the levels of drug effect on a distinct cell or group of cells via probability scoring. This article provides an account for the use of machine learning methods to probe differences in the biophysical, biochemical and physiological characteristics of infected cells in response to pharmacokinetics uptake of drug ingredients for application in cancer, diabetes and neurodegenerative disease therapies. [ABSTRACT FROM AUTHOR]

Ana Casadó,1,2 M Lluïsa Sagristá,1 Margarita Mora1 1Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, Spain; 2Communication and CSR Department, Hospital Clinic of Barcelona, Barcelona, Spain Purpose: Irinotecan (CPT-11) and SN-38 – its active metabolite – are alkaloid-derived topoisomerase I interactive compounds widely used in various cancer therapy protocols. To solve the problems associated with the instability of their lactone ring at physiological pH and with the extreme insolubility of SN-38, the development of delivery carriers (eg, liposomes) has been considered a subject of unquestionable medical interest. This article focuses on the development of an alternative protocol to the classical lipid-film hydration procedures to obtain a pharmaceutical formulation for SN-38.Methods: SN-38-loaded liposomes (SN-38lip) were produced by microemulsification, without a prior lipid-film preparation step, and characterized by different methods. Formulation parameters were determined by photon correlation spectroscopy, and the SN-38 entrapment efficiency was evaluated by absorbance spectroscopy. SN-38lip was obtained as a dry, white powder by lyophilization. MTT and LDH assays were conducted to assess the cytotoxic effect of SN-38, both in liposomal (SN-38lip) and solubilized form (SN-38sol); flow cytometry was used to quantify SN-38 uptake and to analyze cell-cycle phase distribution after drug exposure.Results: Microfluidic, stable, and controlled sized, negatively charged liposomes, with high SN-38 incorporation efficiency into egg yolk phosphatidylcholine (EPC)/L-α-dioleoyl-phospathidylserine (DOPS) (9:1) vesicles (SN-38lip), were prepared. A lyophilized powder of SN-38lip, easily reconstitutable while retaining physicochemical parameters, was finally obtained. The efficacy of SN-38lip was assessed by in vitro studies with two tumor cell lines (HeLa and Caco-2) and compared with that of SN-38sol. It demonstrated the highest uptake of SN-38lip, in accordance with its highest cytotoxicity effect, in comparison with that of SN-38sol. In addition, different cell-cycle alterations were induced in both cell lines by the liposomal formulation.Conclusion: The results highlight the potential usefulness of the procured SN-38 liposomal formulation and provide the basis for conducting in vivo studies that allow the development of alternative strategies for colorectal cancer treatment. Keywords: microfluidic liposomes, drug delivery, SN-38, cytotoxicity, drug uptake, cell-cycle analysis

Although liquid-liquid extraction methods are currently being applied in many areas such as analytical chemistry, biochemical engineering, biochemistry, and biological applications, accessibility and usability of microfluidics in practical daily life fields are still bounded. Suspended microfluidic devices have the potential to lessen the obstacles, but the absence of robust design rules have hampered their usage. The primary objective of this work is to design and fabricate a microfluidic device to quantitatively monitor the drug uptake of cancer cells. Liquid-liquid extraction is used to quantify the drug uptake. In this research work, designs and simulations of two different microfluidic devices for carrying out multiplex solution experiments are proposed to test their efficiency. These simplified miniaturized chips would serve as suspended microfluidic metabolites extraction platform as it allows extracting the metabolites produced from the cancer cells as a result of applying a specific drug type for a certain period of time. These devices would be fabricated by making polydimethylsiloxane (PDMS) molds from the negative master mold using soft lithography. Furthermore, it can leverage to provide versatile functionalities like high throughput screening, cancer cell invasions, protein purification, and small molecules extractions. As per previous studies, PDMS has been depicting better stability with various solvents and has proved to be a reliable and cost effective material to be used for fabrication, though the sensitivity of the chip would be analyzed by cross contamination and of solvents within the channels of device.

Background: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. Methods: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. Results: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. Conclusions: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector. [ABSTRACT FROM AUTHOR]

The intestinal epithelium critically contributes to oral bioavailability of drugs by constituting an important site for drug absorption and metabolism. In particular, intestinal epithelial cells (IEC) actively serve as gatekeepers of drug and nutrient availability. IECs’ transport processes and metabolism are interrelated to the whole-body metabolic state and represent potential points of origin as well as therapeutic targets for a variety of diseases. Human intestinal organoids represent a superior model of the intestinal epithelium, overcoming limitations of currently used in vitro models. Caco-2 cells or rodent explant models face drawbacks such as their cancer and non-human origin, respectively, but are commonly used to study intestinal nutrient absorption, enterocyte metabolism and oral drug bioavailability, despite poorly correlative data. In contrast, intestinal organoids allow investigating distinct aspects of bioavailability including spatial resolution of transport, inter-individual differences and high-throughput screenings. As several countries have already developed strategic roadmaps to phase out animal experiments for regulatory purposes, intestinal organoid culture and organ-on-a-chip technology in combination with in silico approaches are roads to go in the preclinical and regulatory setup and will aid implementing the 3Rs (reduction, refinement and replacement) principle in basic science.

By controlling the passage of small molecules across lipid bilayers, membrane transporters influence not only the uptake and efflux of nutrients, but also the metabolic state of the cell. With more than 450 members, the Solute Carriers (SLCs) are the largest transporter super-family, clustering into families with different substrate specificities and regulatory properties. Cells of different types are, therefore, able to tailor their transporter expression signatures depending on their metabolic requirements, and the physiological importance of these proteins is illustrated by their mis-regulation in a number of disease states. In cancer, transporter expression is heterogeneous, and the SLC family has been shown to facilitate the accumulation of biomass, influence redox homeostasis, and also mediate metabolic crosstalk with other cell types within the tumour microenvironment. This Review explores the roles of membrane transporters in physiological and malignant settings, and how these roles can affect drug response, through either indirect modulation of sensitivity or the direct transport of small-molecule therapeutic compounds into cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Aquaporins (AQPs) are a family of integral membrane channel proteins that facilitate the transport of water and other small solutes across cell membranes. AQPs appear to play crucial roles in parasite survival and represent possible drug targets for novel intervention strategy. In this work, we investigated the tissue distribution and biological roles of an aquaporin TcAQP1 in the neglected parasitic nematode Toxocara canis. Methods: Recombinant C-terminal hydrophilic domain of AQP1 of T. canis (rTcAQP1c) and polyclonal antibody against rTcAQP1c were produced to analyse the tissue expression of native TcAQP1 in adult (female and male) worms using an immunohistochemical approach. RNA interference (RNAi), quantitative real-time PCR (qRT-PCR) and nematocidal assays were performed to investigate the functional roles of TcAQP1 in the adult stage of T. canis. Results: Immunofluorescence analysis showed that TcAQP1 was localised predominantly in the epithelial linings of the reproductive tract and basolateral membrane of the intestine in the adult stage (female and male) of T. canis, indicating important roles in reproduction, nutrient absorption and/or osmoregulation. Treatment with silencing RNA for 24 h resulted in a significant reduction of Tc-aqp-1 mRNA level in adult T. canis, though no phenotypical change was observed. The efficient gene knockdown compromised the nematocidal activity of albendazole in vitro, suggesting the role of TcAQP1 in drug uptake. Conclusions: The findings of this study provide important information about tissue expression and functional roles of TcAQP1 protein in adult T. canis. Understanding the biological functions of this protein in other developmental stages of T. canis and related parasitic nematodes would contribute to the discovery of novel diagnostic or anthelmintic targets. [ABSTRACT FROM AUTHOR]

Abstract The canonical effect of Pregnane X Receptor (PXR, NR1I2) agonism includes enhanced hepatic uptake and a concomitant increase in the first-pass metabolism and efflux of drugs in mammalian liver and intestine. In patients undergoing combination therapy, PXR-mediated gene regulation represents the molecular basis of numerous food-drug, herb-drug, and drug-drug interactions. Moreover, PXR activation promotes chemotherapeutic resistance in certain malignancies. Additional research efforts suggest that sustained PXR activation exacerbates the development of fatty liver disease. Additional metabolic effects of PXR activation in liver are the inhibition of fatty acid oxidation and gluconeogenesis. The identification of non-toxic and selective PXR antagonists is therefore of current research interest. Inhibition of PXR should decrease adverse effects, improve therapeutic effectiveness, and advance clinical outcomes in patients with cancer, fatty liver, and diabetes. This review identifies small molecule PXR antagonists described to date, discusses possible molecular mechanisms of inhibition, and seeks to describe the likely biomedical consequences of the inhibition of this nuclear receptor superfamily member. Highlights • Canonical Pregnane X Receptor (PXR) Function. • PXR Activation and Disease. • Certain Azoles are PXR Antagonists. • Environmental Contaminants and Prescription Medications Inhibit PXR activation. • Natural Products Inhibit PXR Activity. [ABSTRACT FROM AUTHOR]

Abstract: Extraordinary progress in medicinal inorganic chemistry in the past few years led to the rational design of novel platinum compounds, as well as nonplatinum metal-based antitumor agents, including organotin compounds, whose activity is not based on unrepairable interaction with DNA. To overcome poor solubility and toxicity problems that limited the application of these compounds numerous delivering systems were used (Lila et al. in Biol Pharm Bull 37:206-211, 2014; Yue and Cao in Curr Cancer Drug Targets 16:480-488, 2016; Duan et al. in WIREs Nanomed Nanobiotechnol 8:776-791, 2016). Regarding high drug loading capacity, mesoporous silica nanoparticles like SBA-15 became more important for targeted drug delivery. In this study, cellular uptake and biological activities responsible for organotin(IV) compound Ph3Sn(CH2)6OH (Sn6) grafted into (3-chloropropyl)triethoxysilane functionalized SBA-15 (SBA-15p → SBA-15p|Sn6) were evaluated in human melanoma A375 cell line. Moreover, the influence of SBA-15p grafted with organotin(IV) compound on the stemness of A375 cell was tested. Given the fact that SBA-15p|Sn6 nanoparticles are nonspherical and relatively large, their internalization efficiently started even after 15 min with stable adhesion to the cell membrane. After only 2 h of incubation of A375 cells with SBA-15p|Sn6 passive fluid-phase uptake and macropinocytosis were observed. Inside of the cell, treatment with SBA-15p loaded with Sn6 promoted caspase-dependent apoptosis in parallel with senescence development. The subpopulation of cells expressing Schwann-like phenotype arose upon the treatment, while the signaling pathway responsible for maintenance of pluripotency and invasiveness, Wnt, Notch1, and Oct3/4 were modulated towards less aggressive signature. In summary, SBA-15p enhances the efficacy of free Sn6 compound through efficient uptake and well profiled intracellular response followed with decreased stem characteristics of highly invasive A375 melanoma cells. Graphical abstract: [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Glioblastoma is one of the most rapaciously growing cancer within the brain with an average lifespan of 12–15 months (5-year survival <3–4%). Doxorubicin (DOX) is clinically utilized as a first line drug in the treatment of Glioblastoma, however, its restricted entry into the brain via the blood–brain barrier (BBB), limited blood-tumor barrier (BTB) permeability, hemotoxicity, short mean half-life of 1–3 hr as well as rapid body clearance results in tremendously diminished bioactivity in glioblastoma. Dendrimer-Cationized-Albumin (d CatAlb) was synthesized following the carboxyl activation technique and the synthesized biopolymer was characterized by FTIR, MALDI-TOF and zeta potential. The prepared d CatAlb was encrusted on DOX-loaded PLGA nanoparticle core to develop a novel hybrid DOX nanoformulation (d CatAlb-pDNP; particle size: 156 ± 10.85 nm; ƺ: −10.0 ± 2.1 mV surface charge). The formulated d CatAlb-pDNP showed a unique pH-dependent DOX release profile, diminished hemolytic toxicity, higher drug uptake (<0.001) and cytotoxicity in U87MG glioblastoma cells, increase levels of caspase-3 gene in U87MG cells (approximately 5.35-fold higher) inferred that anticancer activity is primarily taking place through caspase-mediated apoptosis mechanism. The developed novel DOX nanoformulation also showed superior trans-epithelial permeation transport across monolayer bEnd.3 cells as well as notable biocompatibility and stability. The d CatAlb-pDNP showed enhanced BBB permeation efficacy as confirmed permeation assay in bEnd.3 cell-based model. The long-term formulation stability of developed nanoformulations was studied by storing them at 5 ± 2 °C and 30 ± 2 °C/60 ± 5% Relative Humidity (% RH) in the stability chamber for a period of 60 days (ICHQ1A (R2)). The outcomes of this investigation evidently indicate that d CatAlb-pDNP offers superior anticancer activity of DOX in glioblastoma cells while significantly improving its BBB permeation. The developed formulation is a biocompatible, safer and commercially viable approach to delivering DOX selectively in sustained manner glioblastoma while countering its hemolytic toxic effect, which is a major ongoing issue with conventional DOX injectable available in the market today. [ABSTRACT FROM AUTHOR]

Organic cation transporters (OCT) 1, 2 and 3 and novel organic cation transporters (OCTN) 1 and 2 of the solute carrier 22 (SLC22) family are involved in the cellular transport of endogenous compounds such as neurotransmitters, l-carnitine and ergothioneine. OCT/Ns have also been implicated in the transport of xenobiotics across various biological barriers, for example biguanides and histamine receptor antagonists. In addition, several drugs used in the treatment of respiratory disorders are cations at physiological pH and potential substrates of OCT/Ns. OCT/Ns may also be associated with the development of chronic lung diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD) and, thus, are possible new drug targets. As part of the Special Issue “Physiology, Biochemistry and Pharmacology of Transporters for Organic Cations”, this review provides an overview of recent findings on the (patho)physiological and pharmacological functions of organic cation transporters in the lung.

• Next-generation antibacterial agents are urgently needed to combat multidrug-resistant, pandrug-resistant gram-negative bacteria and superbugs. • Next generation antibacterial agents are developed by promoting the killing efficacy of first- and second-generation antibacterial agents. • Improved influx of antibacterial agents and delayed development of antibacterial resistance are two key features of next-generation antibacterial agents. • We propose the combination of metabolites with first- or/and second-generation antibacterial agents as a strategy for the development of next-generation antibacterial agents. Existing antibacterial agents can be categorized into two generations, but bacterial insensitivity towards both of these generations poses a serious public health challenge worldwide. Thus, novel approaches and/or novel antibacterials are urgently needed to maintain a concentration of antibacterials that is lethal to bacteria that are resistant to existing antibiotic treatments. Metabolite(s)-based adjuvants that promote antibiotic uptake and enhance antibiotic efficacy are an effective strategy that is unlikely to develop resistance. Thus, we propose a metabolite(s)-based approach, in which metabolites and antibacterials are combined, as a promising strategy for the development of next-generation agents to combat a variety of antibiotic-resistant pathogens. [ABSTRACT FROM AUTHOR]

Biofilm is one of the most important physiological protective barriers of the Streptococcus suis (S. suis), and it is also one of the primary causes of hindrance to drug infiltration, reduction of bactericidal effects, and the development of antibiotic resistance. In order to intervene or eliminate S. suis biofilm, shuttle-shape emodin-loaded nanoparticles were developed in our study. The emodin nanoparticles were prepared by emodin and gelatin–cyclodextrin which was synthesized as drug carrier, and the nanoparticles were 174 nm in size, -4.64 mv in zeta potential, and exhibited a sustained emodin release. Moreover, the delivery kinetics of nanoparticles were also explored in our study. The confocal laser scanning microscopy and colony forming unit enumeration experiment indicated that nanoparticles could increase drug infiltration and uptake by biofilm. The flow cytometry system analysis showed that nanoparticles could be up taken by 99% of the bacteria cells. TCP assay and scanning electron microscopy showed that the nanoparticles had better effect on biofilm inhibition and elimination when compared with emodin solution. These results revealed that the emodin nanoparticles had a better therapeutic effect on the S. suis biofilm in vitro.

Introduction Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a new means of delivering chemotherapy into the abdomen of patients with peritoneal carcinomatosis (PC). The amount of drug uptake in ascites and peritoneum after PIPAC is unknown. Methods Retrospective cohort study of women with PC from gynecological tumors comparing the concentrations of cisplatin and doxorubicin in ascites and peritoneum before and after PIPAC. Concentrations were measured using gas chromatography. Peritoneal tumor samples were assessed for histological tumor regression. Results 59 PIPAC procedures were performed in 32 women with PC. The concentrations of doxorubicin and cisplatin in ascites significantly increased after PIPAC (140.2 ± 671.5 vs 9035.7 ± 5328.6 ng/ml; p < 0.0001 and 95.2 ± 106.4 vs 24,770.8 ± 11,710.8 ng/ml; p < 0.0001, respectively). Concentrations of doxorubicin and cisplatin in peritoneal tissue also significantly increased after PIPAC (5.1 ± 0.7 vs 19.2 ± 38.6 ng/g; p = 0.007, and 81.9 ± 7.8 vs 131.5 ± 134.4 ng/g; p = 0.005, respectively). On an individual patient level, a significant uptake (>2-fold) of doxorubicin and cisplatin was observed in 57/59 (97%) and 58/59 (98%) of cases in ascites and in 23/59 (39%) and 13/59 (22%) of cases in the peritoneum. Uptake of cisplatin and doxorubicin were significantly correlated (Spearman correlation coefficient: 0.33; p = 0.011). After repeated PIPACs, doxorubicin uptake increased in peritoneal tumor tissue (p = 0.008). Conclusions PIPAC leads to a significant chemotherapy uptake in both ascites and peritoneum, suggesting a bimodal cytotoxic effect of PIPAC via direct tissue uptake into peritoneal tumor nodules and via ascites. Consecutive PIPAC applications lead to peritoneal accumulation of doxorubicin, suggesting a cumulative cytotoxic effect of doxorubicin after repeated PIPACs. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the involvement of fatty acid–binding protein 5 (FABP5), a lipid-binding protein expressed at the blood-brain barrier (BBB), in fatty acid and drug uptake into human brain endothelial cells. Following transfection with siRNA against hFABP5, human brain endothelial cell (hCMEC/D3) uptake of lipophilic ligands with varying affinity to FABP5 was assessed with intracellular concentrations quantified by liquid scintillation counting, HPLC, or LCMS/MS. The in situ BBB transport of [ 3 H]-diazepam was also assessed in wild type and FABP5-deficient mice. hFABP5 siRNA reduced FABP5 expression in hCMEC/D3 cells by 39.9 ± 3.8% (mRNA) and 38.8 ± 6.6% (protein; mean ± SEM), leading to a reduction in uptake of [ 14 C]-lauric acid, [ 3 H]-oleic acid, and [ 14 C]-stearic acid by 37.5 ± 8.8%, 41.7 ± 11.6%, and 50.7 ± 13.6%, respectively, over 1 min. No significant changes in [ 14 C]-diazepam, pioglitazone, and troglitazone uptake were detected following FABP5 knockdown in hCMEC/D3 cells. Similarly, no difference in BBB transport of [ 3 H]-diazepam was observed between wild type and FABP5-deficient mice. Therefore, although FABP5 facilitates brain endothelial cell uptake of fatty acids, it has limited effects on brain endothelial cell uptake and BBB transport of drugs with lower affinity for FABP5. [ABSTRACT FROM AUTHOR]

Biofilm is one of the most important physiological protective barriers of the Streptococcus suis (S. suis), and it is also one of the primary causes of hindrance to drug infiltration, reduction of bactericidal effects, and the development of antibiotic resistance. In order to intervene or eliminate S. suis biofilm, shuttle-shape emodinloaded nanoparticles were developed in our study. The emodin nanoparticles were prepared by emodin and gelatin-cyclodextrin which was synthesized as drug carrier, and the nanoparticles were 174 nm in size, -4.64 mv in zeta potential, and exhibited a sustained emodin release. Moreover, the delivery kinetics of nanoparticles were also explored in our study. The confocal laser scanning microscopy and colony forming unit enumeration experiment indicated that nanoparticles could increase drug infiltration and uptake by biofilm. The flow cytometry system analysis showed that nanoparticles could be up taken by 99% of the bacteria cells. TCP assay and scanning electron microscopy showed that the nanoparticles had better effect on biofilm inhibition and elimination when compared with emodin solution. These results revealed that the emodin nanoparticles had a better therapeutic effect on the S. suis biofilm in vitro. [ABSTRACT FROM AUTHOR]

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Although veterinary anthelmintics represent an important source of environmental pollution, the fate of anthelmintics and their effects in plants has not yet been studied sufficiently. The aim of our work was to identify metabolic pathways of the two benzimidazole anthelmintics fenbendazole (FBZ) and flubendazole (FLU) in the ribwort plantain ( Plantago lanceolata L.). Plants cultivated as in vitro regenerants were used for this purpose. The effects of anthelmintics and their biotransformation products on plant oxidative stress parameters were also studied. The obtained results showed that the enzymatic system of the ribwort plantain was able to uptake FLU and FBZ, translocate them in leaves and transform them into several metabolites, particularly glycosides. Overall, 12 FLU and 22 FBZ metabolites were identified in the root, leaf base and leaf top of the plant. Concerning the effects of FLU and FBZ, both anthelmintics in the ribwort plantain cells caused significant increase of proline concentration (up to twice), a well-known stress marker, and significant decrease of superoxide dismutase activity (by 50%). In addition, the activities of four other antioxidant enzymes were significantly changed after either FLU or FBZ exposition. This could indicate a certain risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions. [ABSTRACT FROM AUTHOR]

The search for safe antimalarial compounds acting against asexual symptom-responsible stages and sexual transmission-responsible forms of Plasmodium species is one of the major challenges in malaria elimination programs. So far, among current drugs approved for human use, only primaquine has transmission-blocking activity. The discovery of small molecules targeting different Plasmodium falciparum life stages remains a priority in antimalarial drug research. In this context, several independent studies have recently reported antiplasmodial and transmission-blocking activities of commonly used stains, dyes and fluorescent probes against P. falciparum including chloroquine-resistant isolates. Herein we have studied the antimalarial activities of dyes with different scaffold and we report that the triarylmethane dye (TRAM) Brilliant green inhibits the growth of asexual stages (IC 50 ≤ 2 μM) and has exflagellation-blocking activity (IC 50 ≤ 800 nM) against P. falciparum reference strains (3D7, 7G8) and chloroquine-resistant clinical isolate (Q206). In a second step we have investigated the antiplasmodial activities of two polysulfonated triarylmethane food dyes. Green S (E142) is weakly active against P. falciparum asexual stage (IC 50 ≃ 17 μM) whereas Patent Blue V (E131) is inactive in both antimalarial assays. By applying liquid chromatography techniques for the culture supernatant analysis after cell washings and lysis, we report the detection of Brilliant green in erythrocytes, the selective uptake of Green S (E142) by infected erythrocytes, whereas Patent Blue V (E131) could not be detected within non-infected and 3D7-infected erythrocytes. Overall, our results suggest that two polysulfonated food dyes might display different affinity with transporters or channels on infected RBC membrane. [ABSTRACT FROM AUTHOR]

Irinotecan (CPT-11) is an effective chemotherapeutic agent widely used to treat different cancers. Otherwise, the liposomal delivery of anti-tumor agents has been shown to be a promising strategy. The aim of this study has been to analyze the effect of liposomal CPT-11 (CPT-11lip) on two human cell lines (Hs68 and HeLa) to establish the suitability of this CPT-11 nanocarrier. We have demonstrated the highest uptake of CPT-11lip in comparison with that of CPT-11sol, in lactate buffer, and that CPT-11lip was internalized in the cells through an endocytic process whereas CPT-11sol does so by passive diffusion. CPT-11lip was not cytotoxic to normal fibroblast Hs68 cells, but induced a massive apoptosis accompanied by cell senescence in HeLa cells. CPT-11lip treatment modified the morphology of HeLa cells, induced different cell cycle alterations and accumulated into lysosomes in both cell lines. In particular, CPT-11lip treatment showed that surviving HeLa cells remained in a state of senescence whereas only a temporal growth arrest was induced in Hs68 cells. Results of RT-PCR indicated that the different responses in Hs68 (survival) and HeLa cells (apoptotic death), seemed to be induced by a p53- and p53- independent mechanism, respectively. An analysis of DNA damage also determined that released CPT-11 from liposomes was able to reach the nucleus and exert a genotoxic effect in both cell lines, which was repaired in Hs68 but not in HeLa cells. All results indicate that phospholipid-cholesterol liposomes possess optimum properties for CPT-11 delivery, being biocompatible and selectively cytotoxic against HeLa tumorigenic cells. [ABSTRACT FROM AUTHOR]

An analytical procedure to measure the whole-body uptake of pharmaceuticals in zebrafish has been developed using state-of-the-art methodologies. A sample preparation procedure for 9 pharmaceuticals displaying a variety in physicochemical properties was developed using 10-day old zebrafish (TG898). For an efficient homogenization of the samples and subsequent recovery of the compounds of interest, different amounts of organic solvents in combination with acidic modifiers were added to zebrafish samples. Samples were subsequently processed using a powerful bath sonicator and centrifuged. Supernatant was then removed and evaporated in a vacuum oven before being reconstituted in a mobile phase-like solvent. Samples were analyzed using ultra-high performance liquid chromatography (UHPLC) on an Acquity BEH C 18 column (100 × 2.1 mm, d p =1.7 µm) coupled to a Waters Xevo TQ-S mass spectrometer. For this purpose, a generic gradient was run, wherein the percentage of acetonitrile was varied from 3% to 82% in 10.5 min at a flow rate of 0.41 mL/min. Linearity of the method was demonstrated for all compounds (R 2 > 0.997) in a practically relevant concentration range. Matrix effects were between 81% and 106%, except for amitriptyline (51%). Using this method, it was demonstrated that a sample pretreatment using 1:2 (v/v) water:methanol in combination with 0.1% formic acid resulted in acceptable recoveries between 74% and 100% for all compounds. Together with the obtained lower limits of quantification of the analytical method (between 0.005 and 1.5 ng/mL), this allowed the use of a single zebrafish to study the whole-body uptake of a particular drug, after incubating zebrafish at the maximum tolerated concentration for this drug. [ABSTRACT FROM AUTHOR]