Development and optimization of a new processing approach for manufacturing topical liposomes-in-hydrogel drug formulations by dual asymmetric centrifugation

Objective: The objective of the present study was to utilize dual asymmetric centrifugation (DAC) as a novel processing approach for the production of liposomes-in-hydrogel formulations.

Materials and methods: Lipid films of phosphatidylcholine, with and without chloramphenicol (CAM), were hydrated and homogenized by DAC to produce liposomes in the form of vesicular phospholipid gels with a diameter in the size range of 200?300?nm suitable for drug delivery to the skin. Different homogenization processing parameters were investigated along with the effect of adding propylene glycol (PG) to the formulations prior to homogenization. The produced liposomes were incorporated into a hydrogel made of 2.5% (v/v) soluble β-1,3/1,6-glucan (SBG) and mixed by DAC to achieve a homogenous liposomes-in-hydrogel-formulation suitable for topical application.

Results and discussion: CAM-containing liposomes with a vesicle diameter of 282?±?30?nm and polydispersity index (PI) of 0.13?±?0.02 were successfully produced by DAC after 50?min centrifugation at 3500?rpm, and homogenously (Conclusion: We managed to develop a relatively fast and reproducible new method for the production of liposomes-in-hydrogel formulations by DAC.     

Genistein affects ovarian folliculogenesis: A stereological study

The effects of short‐term genistein exposure on ovarian folliculogenesis in immature rats were examined stereologically. To determine whether genistein acts as an estrogen agonist or antagonist, the results were compared with the effects of 17α‐ethynylestradiol. Immature female rats received 50 mg/kg/bw of genistein in dimethyl sulfoxide subcutaneously daily for three consecutive days from 18 to 20 days. The second group was injected with 1 μg/kg/bw of 17α‐ethynylestradiol in olive oil in the same schedule. Each group had a corresponding control. Genistein increased ovary and ovarian stroma volumes by 18.50% (P < 0.05) and 53.40% (P < 0.05), respectively, and changed the parenchyma to stroma ratio in favor of stroma. Genistein induced decreases in the number of primordial (by 17.23%; P < 0.05), primary (16.62%; P < 0.05), and secondary follicles (12.29%: P < 0.05), whereas the number of atretic secondary follicles increased (5.10‐fold; P < 0.05). The number of healthy large follicles was raised by 27.3% (P < 0.05), accompanied by 35.64% more atretic large follicles (P < 0.05). Similarly to genistein, estradiol changed the parenchyma to stroma ratio in favor of stroma, and reduced the number of primordial follicles, but the number of primary follicles was elevated. There were more healthy and atretic small and large follicles. In conclusion, genistein acted as an estrogen antagonist and had an inhibitory effect on the initial phase of folliculogenesis. In the other phases, genistein acted as an estrogen agonist, stimulating transition from the preantral to antral stage of folliculogenesis, and altering the ratio of follicular parenchyma and ovarian stroma in favor of stroma. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Binuclear metallohydrolases: complex mechanistic strategies for a simple chemical reaction

Binuclear metallohydrolases are a large family of enzymes that require two closely spaced transition metal ions to carry out a plethora of hydrolytic reactions. Representatives include purple acid phosphatases (PAPs), enzymes that play a role in bone metabolism and are the only member of this family with a heterovalent binuclear center in the active form (Fe(3+)-M(2+), M = Fe, Zn, Mn). Other members of this family are urease, which contains a di-Ni(2+) center and catalyzes the breakdown of urea, arginase, which contains a di-Mn(2+) center and catalyzes the final step in the urea cycle, and the metallo-β-lactamases, which contain a di-Zn(2+) center and are virulence factors contributing to the spread of antibiotic-resistant pathogens. Binuclear metallohydrolases catalyze numerous vital reactions and are potential targets of drugs against a wide variety of human disorders including osteoporosis, various cancers, antibiotic resistance, and erectile dysfunctions. These enzymes also tend to catalyze more than one reaction. An example is an organophosphate (OP)-degrading enzyme from Enterobacter aerogenes (GpdQ). Although GpdQ is part of a pathway that is used by bacteria to degrade glycerolphosphoesters, it hydrolyzes a variety of other phosphodiesters and displays low levels of activity against phosphomono- and triesters. Such a promiscuous nature may have assisted the apparent recent evolution of some binuclear metallohydrolases to deal with situations created by human intervention such as OP pesticides in the environment. OP pesticides were first used approximately 70 years ago, and therefore the enzymes that bacteria use to degrade them must have evolved very quickly on the evolutionary time scale. The promiscuous nature of enzymes such as GpdQ makes them ideal candidates for the application of directed evolution to produce new enzymes that can be used in bioremediation and against chemical warfare. In this Account, we review the mechanisms employed by binuclear metallohydrolases and use PAP, the OP-degrading enzyme from Agrobacterium radiobacter (OPDA), and GpdQ as representative systems because they illustrate both the diversity and similarity of the reactions catalyzed by this family of enzymes. The majority of binuclear metallohydrolases utilize metal ion-activated water molecules as nucleophiles to initiate hydrolysis, while some, such as alkaline phosphatase, employ an intrinsic polar amino acid. Here we only focus on catalytic strategies applied by the former group.     

Unbiased stereological estimation of the rat fetal pituitary volume and of the total number and volume of TSH cells after maternal dexamethasone application

Glucocorticoids have an inhibitory influence on proliferation activity of the pituitary cells while stimulating apoptosis. Therefore, it was hypothesized that the synthetic glucocorticoid, dexamethasone (DX), has an inhibitory influence on the number of thyroid‐stimulating hormone (TSH) cells during fetal development. The effects of maternal administration of DX on stereological parameters of TSH cells, and TSH serum concentration were investigated in 21‐day‐old rat fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg DX/kg b.w. subcutaneously, followed by 0.5 mg DX/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline vehicle. TSH cells were stained immunocytochemically by the peroxidase–antiperoxidase (PAP) method. The fetal pituitary volumes were estimated using Cavalieri's principle. A physical disector counting technique in combination with the fractionator sampling method was used for estimation of pituitary TSH cell number. Cell and nuclear volumes were measured with a planar rotator. Maternal DX application was found to cause a significant decrease of pituitary volume and number of TSH cells per pituitary in 21‐day‐old fetuses in comparison with the control fetuses. TSH cell number expressed per body weight unit declined significantly after maternal DX administration. These results indicate an inhibitory DX influence on proliferative activity of precursors and likely differentiated TSH cells and increased apoptotic prevalence. The histological appearance, volume of TSH cells and TSH serum concentration suggest intensive synthetic activity in TSH cells of DX exposed fetuses. Microsc. Res. Tech. 73:1077–1085, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis and structural investigations on aluminium-free Ti-Beta/SBA-15 composite

An aluminium-free Ti-Beta/SBA-15 composite material was prepared by the post-synthesis incipient wetness-deposition of different amounts of Ti-Beta nanoparticles solution on the SBA-15 matrix. The presence of crystalline nanoparticles in the solution, used for impregnation on SBA-15, was confirmed by HRTEM measurements. The hexagonal arrangement of the mesopores of SBA-15 was proven by XRD and TEM measurements. The presence and the deposition of Ti-incorporated zeolitic nanoparticles on the mesopore walls of SBA-15 were proven by nitrogen sorption analysis, IR spectroscopy and TG analysis. The X-ray absorption spectroscopy analysis of local environment of titanium incorporated in the new composite material showed that the product contained five fold coordinated framework titanium. These Ti sites can be oxidation titanium sites.     

Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.     

Sorption of textile dye from aqueous solution by macroporous amino‐functionalized copolymer

Macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) (PGME) was synthesized by suspension copolymerization and functionalized with diethylene triamine (PGME‐deta). The effect of pH, contact time, and sorbent mass on sorption efficiency of initial and functionalized copolymer sample for removal of Acid Orange 10 dye from aqueous solutions was studied. No dye was sorbed by nonfunctionalized copolymer, indicating that sorption of Acid Orange 10 by PGME‐deta is specific, through amino groups. The isotherm data are best fitted by Langmuir model, indicating homogeneous distribution of active sites in PGME‐deta as well as monolayer sorption. Sorption kinetics study showed that the sorption of Acid Orange 10 by PGME‐deta obeys the pseudo‐second‐order kinetic model. It was shown that PGME‐deta selectively sorbs Acid Orange 10 from binary solution with Bezaktiv Rot reactive dye. The comparison of sorption characteristics of PGME‐deta with activated carbon showed that this functionalized copolymer might be used as an alternative sorbent for textile dyes. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Interaction between Dipolar Lipid Headgroups and Charged Nanoparticles Mediated by Water Dipoles and Ions

In this work, a theoretical model describing the interaction between a positively or negatively charged nanoparticle and neutral zwitterionic lipid bilayers is presented. It is shown that in the close vicinity of the positively charged nanoparticle, the zwitterionic lipid headgroups are less extended in the direction perpendicular to the membrane surface, while in the vicinity of the negatively charged nanoparticle, the headgroups are more extended. This result coincides with the calculated increase in the osmotic pressure between the zwitterionic lipid surface and positively charged nanoparticle and the decrease of osmotic pressure between the zwitterionic lipid surface and the negatively charged nanoparticle. Our theoretical predictions agree well with the experimentally determined fluidity of a lipid bilayer membrane in contact with positively or negatively charged nanoparticles. The prospective significance of the present work is mainly to contribute to better understanding of the interactions of charged nanoparticles with a zwitterionic lipid bilayer, which may be important in the efficient design of the lipid/nanoparticle nanostructures (like liposomes with encapsulated nanoparticles), which have diverse biomedical applications, including targeted therapy (drug delivery) and imaging of cancer cells.