A Reversed-phase High Performance Liquid Chromatography (HPLC) method for bio-analysis of Methotrexate

Methotrexate (MTX) is a chemotherapeutic agent used in treatment of many disorders including autoimmune diseases and cancers. The availability of a reliable analysis method for drug assay in biological fluids of interest  is  a  prerequisite  for  all  pharmacokinetic  studies  in  humans  or  animal  models. Considering the complex matrices of the biological specimens as well as the low concentrations of the majority of the drugs in biological fluids, the development of an available while sensitive method for the bioanalytical studies is often a challenging issue.For drug assay in aqueous, plasma, animal brain and liver tissue environments in a concentration range of 25-600 ng/ml, a reverse phase high performance liquid chromatography (RP-HPLC) was developed.System suitability tests were indicating a method with acceptable analytic separation efficiency and peak shape proving method’s selectivity. Limit of detection (LOD) and limit of quantification (LOQ) determined to be 10 ng/ml and 25ng/ml, which reflect method sensitivity. Regression analysis showed a linear correlation between area under curve (AUC) of peaks and corresponding MTX concentrations. The within-day and between-day precision and accuracy was both in acceptable ranges. Recovery index of method for median concentration (200 ng/ml) is also about 74%.The developed method was accorded to the acceptable criteria of analytical method validation. The sensitivity of the method in all the tested matrices made the method suitable in terms of detection and quantitation of low concentration samples throughout the study. Also, the assay method had fairly short run-time and lacks any significant interference. </p

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics

Bacterial extracellular vesicles: Understanding biology promotes applications as nanopharmaceuticals.

Extracellular vesicle (EV)-mediated communication between proximal and distant cells is a highly conserved characteristic in all of the life domains, including bacteria. These vesicles that contain a variety of biomolecules, such as proteins, lipids, nucleic acids, and small-molecule metabolites play a key role in the biology of bacteria. They are one of the key underlying mechanisms behind harmful or beneficial effects of many pathogenic, symbiont, and probiotic bacteria. These nanoscale EVs mediate extensive crosstalk with mammalian cells and deliver their cargos to the host. They are stable in physiological condition, can encapsulate diverse biomolecules and nanoparticles, and their surface could be engineered with available technologies. Based on favorable characteristics of bacterial vesicles, they can be harnessed for designing a diverse range of therapeutics and diagnostics for treatment of disorders including tumors and resistant infections. However, technical limitations for their production, purification, and characterization must be addressed in future studies

in vitro- and in vivo Evaluation of Methotrexate-Loaded Hydrogel Nanoparticles Intended to Treat Primary CNS Lymphoma via Intranasal Administration

Purpose: Although it passes through blood-brain barrier (BBB) very poorly, methotrexate (MTX) is an important therapeutic in the treatment of many central nervous system malignancies. Accordingly, intranasal (IN) administration accompanied with a muco-adhesive chitosan-based nanoformulation is expected to overcome this problem. Methods: Nanogel containing MTX was prepared through an ionic gelation method and then characterized in terms of particle size, morphology, zeta potential, drug loading and drug release behavior. The drug release results were fitted on eight mathematical models to choose the model best describing the phenomenon. Then the nano-formulation and free drug solution in deionized water as control were administered in the nasal cavity for rats and after 15, 30, 60 and 240 minutes their brain and plasma were analyzed for MTX quantity. Results: The nano-formulation demonstrated an average particle size near 100 nm with a zeta potential of 18.65±1.77 mv. Loading efficiency and loading capacity were calculated to be 65.46±7.66 and 3.02±0.34 respectively. The Weibull model was found to be best describing the release phenomenon as a combination of swelling and Fickian diffusion. Moreover in in vivo studies, drug targeting efficiency and direct transport percentage for nanogel (test) and free drug solution (control) were 424.88% and 76.46% and 34842.15% and 99.71% respectively.  Conclusion: According to in vivo studies, nanogel produced significantly higher concentration of MTX in the brain but not in the plasma when compared to the free drug solution. Besides, in comparison to intravenous administration of the same nanogel it was indicated that intranasal administration significantly increases the brain concentration of MTX

Chemically Engineered Immune Cell-Derived Microrobots and Biomimetic Nanoparticles : Emerging Biodiagnostic and Therapeutic Tools

Over the past decades, considerable attention has been dedicated to the exploitation of diverse immune cells as therapeutic and/or diagnostic cell-based microrobots for hard-to-treat disorders. To date, a plethora of therapeutics based on alive immune cells, surface-engineered immune cells, immunocytes' cell membranes, leukocyte-derived extracellular vesicles or exosomes, and artificial immune cells have been investigated and a few have been introduced into the market. These systems take advantage of the unique characteristics and functions of immune cells, including their presence in circulating blood and various tissues, complex crosstalk properties, high affinity to different self and foreign markers, unique potential of their on-demand navigation and activity, production of a variety of chemokines/cytokines, as well as being cytotoxic in particular conditions. Here, the latest progress in the development of engineered therapeutics and diagnostics inspired by immune cells to ameliorate cancer, inflammatory conditions, autoimmune diseases, neurodegenerative disorders, cardiovascular complications, and infectious diseases is reviewed, and finally, the perspective for their clinical application is delineated.Peer reviewe

Cell-Derived Vesicles for Antibiotic Delivery—Understanding the Challenges of a Biogenic Carrier System

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics