Preparation of novel shell-ionotropically crosslinked micelles based on hexadecylamine and tripolyphosphate for cancer drug delivery.

AIMS: Micellar systems have the advantage of being easily prepared, cheap, and readily loadable with bioactive molecular cargo. However, their fundamental pitfall is poor stability, particularly under dilution conditions. We propose to use simple quaternary ammonium surfactants, namely, hexadecylamine (HDA) and hexadecylpyridinium (HDAP), together with tripolyphosphate (TPP) anion, to generate ionotropically stabilized micelles capable of drug delivery into cancer cells. METHODS: optimized mixed HDA/HDAP micelles were prepared and stabilized with TPP. Curcumin was used as a loaded model drug. The prepared nanoparticles were characterized by dynamic light scattering, infrared spectroscopy, transmission electron microscopy, and differential scanning calorimetry. Moreover, their cellular uptake was assessed using flow cytometry and confocal fluorescence microscopy. RESULTS: The prepared nanoparticles were found to be stable under dilution and at high temperatures and to have a size range from 139 nm to 580 nm, depending on pH (4.6-7.4), dilution (up to 100 times), and temperature (25 - 80 °C). They were effective at delivering their load into cancer cells. Additionally, flow cytometry indicated the resulting stabilized micellar nanoparticles to be non-cytotoxic. CONCLUSIONS: The described novel stabilized micelles are simple to prepare and viable for cancer delivery.

Functional and morphological study of the effects of carvacrol on smooth muscle of the thoracic aorta in the rat.

Background: The leaves of Origanum are widely used in herbal medicine hence of having many beneficial ingredients, one of these important compounds is Carvacrol. The inhibitory effect of Carvacrol was the core of this study by applying different kinds of stimulants to smooth muscles in the wall of thoracic aorta in rats. Aim: To investigate the pharmacological effects of Carvacrol, the main active ingredient present in the medicinal plant Origanum, on the contractile activity and morphology of the smooth muscle of the rat thoracic aorta. Materials and Methods: After the thoracic aorta arteries were isolated and prepared for the experiments, each thoracic aorta was cut into 5-mm ring segments; different stimulants were used (Potassium Chloride, Norepinephrine, U46619, and α,ß-methylene ATP) in the presence and absence of Carvacrol on four groups of rats. The isolated rings were placed and connected to a force transducer which in turn linked to a data acquisition system via an amplifier to record the effect of each stimulant. GraphPad Prism version 5.02 for Windows, one-way analysis of variance followed by Dunnett's multiple comparison test. Results: It was found out that Carvacrol obstructs the contractile responses elicited by exogenous NA, KCl, U46619, and α,ß-methylene ATP in a concentration dependent manner. Conclusion: The addition of Carvacrol in the experimental rats showed an increase in the thickness of tunica media as evident by the number of smooth muscle layers and laminae of elastic fibers. It was found that Carvacrol reduced the vascular smooth muscle contractility in the rat thoracic aorta. The mechanism of action is presumed to be achieved through interfering with the mobilization of both intracellular and extracellular Ca2+ through different receptors. Furthermore, it might be suggested that Carvacrol in high doses stimulates smooth muscles in the wall of aorta leading to an increase in the thickness of tunica media layer.

Assessment of Proteolysis by Pyrylium and Other Fluorogenic Reagents.

AIMS: We aim to evaluate the potential application of amine reactive fluorogenic reagents for estimating enzymatic proteolysis. BACKGROUND: Proteolytic enzymes play important roles in regulating many physiological processes in living organisms. OBJECTIVES: Assessment of protein degradation by using reagents for protein assay techniques. METHODS: We have assayed samples at the start and after 30-60 minutes incubation with trypsin by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents and NanoOrange as non-amine reactive dye. RESULTS: All BSA prepared samples with trypsin have shown significantly higher fluorescence intensity (FI) versus controls (which reflects proteolysis) when assayed by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents. However, same samples assayed with NanoOrange as non-amine reactive reagent did not show any significant variation between samples containing trypsin and controls. CONCLUSION: These results are confirming reliability of highly sensitive protein assays utilizing amine reactive fluorogenic reagents for general estimation of proteolysis.

Two-Color Analysis of Leukocytes Labeled by Modified RBCs and Their Fragments.

Red blood cells (RBCs) are attractive tools for surface modification to adhere specifically to molecules, cellular fragments (e.g., microvesicles), or whole cells for potential use in bioanalytical assays or as a delivery vehicle in targeted therapy. Within this study, we have loaded RBCs with fluorochrome-conjugated antibodies (Ab) against CD45 and CD22 leukocyte markers and evaluated the conjugation process by microscopy. We have assessed the potential application of RBCs fragments generated from conjugated RBCs for targeting Cyto-Trol control cells by flow cytometric (FCM) approaches. Based on their scattering and fluorescence characteristics (FITC and PE expression), modified RBCs and their fragments, Cyto-Trol cells, and clusters of both were distinguished by two color FCM analysis. Fragments with anti-human Kallestad Ab as a nonspecific FITC conjugate had less than 20% binding to Cyto-Trol controls compared to CD45-FITC Ab conjugate with nearly 100% binding capacity. Cyto-Trol-microvesicle-clusters were more than 45% positive for either FITC or PE. Anti-CD22-PE modified RBCs fragments were also useful in staining and showing about 19.5% positively stained events in the Cyto-Trol region. The proof-of-concept shows, that specific antibody can be attached to RBCs, and generated fragments can be useful to stain target cells for FCM analysis. © 2018 International Society for Advancement of Cytometry.

Pharmacokinetic evaluation of D-ribose after oral and intravenous administration to healthy rabbits.

INTRODUCTION: This study explored D-ribose pharmacokinetics after intravenous (IV) and oral administration to healthy rabbits. MATERIALS AND METHODS: D-ribose was administered once as 420 mg/kg (N=4) or 840 mg/kg (N=6) dose intravenously, or as an oral dose of 420 mg/kg (N=3) or 840 mg/kg (N=3). Serum was obtained at various time points, up to 210 minutes after administration. Urine was also collected after IV administration. Pharmacokinetic parameters were determined from drug concentration-time data using Kinetica software. RESULTS: The findings showed that D-ribose follows a dose-dependent kinetic profile. With doubling the IV dose, AUCtotal was significantly increased by threefold, while the clearance was decreased by 44%. The half-life was 1.7-fold longer at the higher dose. Similar nonsignificant trends were also observed at oral administration. D-ribose was rapidly absorbed (Tmax=36-44 minutes) and rapidly disappeared from plasma (within <140 minutes). Additionally, D-ribose was partially (18-37.5%) recovered from urine. CONCLUSION: Collectively, D-ribose showed a dose-dependent kinetic profile, where parameters change according to dosing levels. D-ribose clearance seems to follow first-order kinetics at low dose. Thereafter, elimination systems are saturated, and elimination continues in a fast manner. Urine recovery was partial, which could be attributed to the several metabolic pathways that pentose can undergo.

Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications.

This study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4',6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

Prevalence of vitamin-mineral supplement use among Jordan University students.

OBJECTIVE: To determine the prevalence, reasons, source of information of vitamin-mineral supplement uses, and their relationship with selected demographic and lifestyle characteristics among Jordan University students. METHODS: A cross-sectional survey was carried out at Jordan University, Amman, Jordan from March to September 2005. The survey included 1,187 students aged 17-28 years. A multistage stratified sampling technique was used to recruit the participants from different majors at Jordan University. A self-administered questionnaire containing questions on use of vitamin-mineral supplement during the last year, demographic, and lifestyle characteristics was completed. RESULTS: The overall prevalence of vitamin-mineral supplement use during the last year reported by students was 27.4% (males 22%, and females 30.2%). Vitamin-mineral supplement use among university students was significantly (p<0.05) associated with age, gender, family monthly income, smoking status, physical activity, vegetarian status, and body mass index. The most frequently used supplements were multivitamins (10.4%) and multivitamins-multi minerals (10%). The most frequently given reason for supplement use was for treatment. The main frequently reported source of information was a physician (45.8%). CONCLUSION: The overall prevalence rate of vitamin-mineral supplement use is very low. Supplement users are more likely to be: females, nonsmokers, of higher income, physically active, vegetarians, and of normal body mass index. The main reason for supplement use is for treatment, and the major source of information on supplements is by physicians and pharmacists.