Emir Baki Denkbaş

Regulation of Tumor Angiogenesis by EZH2

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Magnetic chitosan microspheres: preparation and characterization

Abstract In this study, magnetic chitosan microspheres were prepared in a well shaped spherical form with a size range of 100 to 250 μm (size distribution ±15 to ±40 μm, respectively) by the suspension cross-linking technique for use in the application of magnetic carrier technology. The magnetic material (i.e. Fe3O4) used in the preparation of the magnetic chitosan microspheres was prepared by precipitation from FeSO4 and Fe2(SO4)3 solutions in basic medium and then ground to the desired size (i.e. 1–5 μm). The morphological and magnetic properties of the microspheres were characterized by different techniques (i.e. SEM, optical microscopy, magnetometry). The results demonstrated that the stirring rate of the suspension medium and the Fe3O4/chitosan ratio are the most effective parameters for the size/size distribution and the magnetic quality of the microspheres, while the chitosan molecular weight (MW) has no significant effect on these properties for the given MW range (i.e. 150 to 650 kDa). The best magnetic quality of the magnetic chitosan microspheres is around 9.1 emu/g microsphere at 10 kG magnetic field intensity.

Perspectives on: Chitosan Drug Delivery Systems Based on their Geometries:

Chitosan is a natural polymer that has many physicochemical (polycationic, reactive OH and NH2 groups) and biological (bioactive, biocompatible, biodegradable) properties. These unique properties make chitosan an excellent material for the development of new biomedical applications. One of the most well known biomedical chitosan applications is in drug delivery systems. Chitosans have been used in the design of many different types of drug carriers for various administration routes such as oral, bucal, nasal, transdermal, parenteral, vaginal, cervical, intrauterine and rectal. Chitosan can be engineered into different shapes and geometries such as nanoparticles, microspheres, membranes, sponges and rods. This paper is a perspective on the preparation of the chitosan drug delivery systems based on different structural geometries. In this respect, special preparation techniques are used to prepare chitosan drug carriers by altering such parameters as crosslinker concentration, chitosan molecular weight, drug/polymer ratio and processing conditions all of which affect the morphology of chitosan drug carriers and release rate of the loaded drug.

Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies

Dry eye syndrome is a common disorder of the tear film caused by decreased tear production or increased evaporation. The objective of this study was to evaluate the potential effectiveness of Cyclosporine A (CsA) nanoparticles (NPs) for the treatment of inflammation of the eye surface. Topical CsA is currently the only and safe pharmacologic treatment of severe dry eye symptoms. The NPs were prepared using either poly-lactide-co-glycolide (PLGA) or a mixture of PLGA with Eudragit®RL or were coated with Carbopol®. The mean size of CsA loaded NPs was within the range from 148 to 219nm, except for the Carbopol® coated NPs (393nm). The drug entrapment efficiency was very high (from 83 to 95%) and production yield was found between 75 and 92% in all preparations. The zeta potential of the Eudragit® RL containing NPs was positive (19-25mV). The NPs formulations exhibited a biphasic drug release with initial burst followed by a very slow drug release and total cumulative release within 24h ranged from 75 to 90%. Kinetically, the release profiles of CsA from NPs appeared to fit best with the Weibull model. The viability of L929 cells was decreased by increasing the concentration of the various NPs examined as well as the incubation time. The amount of NPs uptake was related to the polymer type used. The highest degree of cellular uptake (52.2%), tear film concentration of the drug (366.3ng/g) and AUC(0→24) (972.6ngh/g) value were obtained from PLGA: Eudragit® RL (75:25)-CsA NPs formulations. The change of surface characteristics of NPs represents a useful approach for improvement of ocular retention and drug availability.

Chitosan microspheres and sponges: Preparation and characterization

In this study, chitosan microspheres and sponges were prepared and characterized for diverse biomedical applications successfully. The chitosan microspheres were obtained with a “suspension crosslinking technique” in the size range of 30–700 μm. The stirring rate of the suspension medium and the chitosan/acetic acid ratio, emulsifier, and crosslinker, that is, the glutaraldehyde concentration in the suspension medium, were evaluated as the effective parameters on the size/size distributions of the microspheres. The microsphere size/size distributions were increased with the decreasing of all effective parameters except the chitosan/acetic acid ratio. In the second part of the study, chitosan sponges were prepared with a solvent-evaporation technique and sponges were cross-linked either during the formation or after the formation of sponges by using a cross-linker, that is, glutaraldehyde. When the sponges were crosslinked during the formation, fibrillar structures were obtained, while the leaflet structures were obtained in the case of crosslinking after the formation of sponges. In the last part of the study, the swelling behavior of both the chitosan microspheres and sponges were evaluated using different amounts of the crosslinker. The swelling ratio was increased in both types of structures, that is, microspheres and sponges, by decreasing the amount of the crosslinker.

Rifampicin carrying polyhydroxybutyrate microspheres as a potential chemoembolization agent.

In this study, we attempted to prepare microspheres from a microbial biodegradable polyester, i.e. polyhydroxybutyrate (PHB) as a potential chemoembolization agent. The drug loaded PHB microspheres were prepared by a solvent evaporation technique, in which methylene chloride, distilled water, and polyvinyl alcohol were utilized as the solvent, dispersion medium, and emulsifier, respectively. Microspheres were obtained within a size range of 5-100 microns by changing the initial polymer/solvent ratio, emulsifier concentration, stirring rate, and initial drug concentration. It was possible to obtain PHB with very narrow size distributions by applying gravity field-flow fractionation technique. Very high drug loadings of up to 407.6 mg rifampicin/g PHB were achieved. Drug release rates were very rapid. Almost 90% of the drug loaded was released in about 24 h. Both the size and drug content of PHB microspheres were found to be effective in controlling the drug release from these microspheres.

5-fluorouracil loaded chitosan microspheres for chemoembolization.

In this study, chitosan microspheres were prepared by a suspension cross-linking technique. A petroleum ether/mineral oil mixture was used as the suspension medium which includes an emulsifier, e.g. Tween-80. Glutaraldehyde was used as the cross-linker. 5-Fluorouracil was incorporated in the matrix for the possible use of the microspheres in chemoembolization. The size and size distribution of the chitosan microspheres varied in the size range of 100-200 microns, by changing the emulsifier concentration, stirring rate, chitosan/solvent ratio and drug/chitosan solution ratio. In summary, the size and size distribution of the microspheres decreased when the emulsifier concentration and stirring rate were increased. Smaller microspheres with narrower size distributions were obtained when the chitosan/solvent ratio and drug/chitosan ratio were lower. It was possible to load the chitosan microspheres with 5-FU to a concentration of 10.4 mg 5-FU/g chitosan. Around 60% of the loaded drug was released within the first 24 h, then the release rate became much slower.

The use of polyethyleneglycolmethacrylate-co-vinylimidazole (PEGMA-co-VI) microspheres for the removal of nickel(II) and chromium(VI) ions

The polyethyleneglycolmethacrylate-co-vinylimidazole (PEGMA-VI) copolymers, that can be used in heavy metal removal applications, were synthesized and characterized; and their use as sorbents in heavy metal removal was investigated. It was determined that the ligand vinylimidazole was successfully inserted into the polymer structure. Then, chromium (Cr(VI)) and nickel (Ni(II)) ions were used as model species to investigate the usability of the obtained microspheres in heavy metal removal. The effects of pH of the adsorption medium, initial concentration of the metal ions and VI content of PEGMA-VI microspheres were investigated as the effective parameters on the adsorption capacities of the microspheres. The adsorption rate of the microspheres was also investigated for determination of the optimum adsorption time which is the required time for maximum adsorption capacity. The adsorption capacities under optimum conditions were also determined. The order of adsorption affinities of PEGMA-VI microspheres with respect to the used metals was determined by competitive adsorption studies. According to the obtained results, the highest adsorption affinity of the PEGMA-VI microspheres was towards Cr(VI) ions, the adsorption affinity was less for Ni(II) and the least affinity was towards Cu(II) ions. The adsorption-desorption studies showed that the microspheres were reusable without a significant decrease in the ion adsorption capacities.

Preparation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHX) based nanoparticles for targeted cancer therapy

Targeted drug delivery systems are one of the most promising alternatives for the cancer therapy. Rapid developments on nanomedicine facilitated the creation of novel nanotherapeutics by using different nanomaterials. Especially polymer based nanoparticles are convenient for this purpose. In this study; a natural polymer (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHBHHX) was used as a base matrix for the production of a novel nanotherapeutic including antineoplastic agent, Etoposide and attached folic acid as a ligand on the nanoparticles. Modified solvent evaporation technique was used for the production of PHBHHX nanoparticles and the average size of the obtained PHBHHX nanoparticles were observed in the range of 180 nm and 1.5 μm by the change in experimental conditions (i.e., homogenization rate, surfactant concentration and polymer/solvent ratio). By the increase in homogenization rate and surfactant concentration, size of the nanoparticles was decreased, while the size was increased by the increase in polymer/solvent ratio. Drug loading ratio was also found to be highly affected by polymer/drug ratio. Surface charge of the prepared nanoparticles was also investigated by zeta potential measurements. In the cytotoxicity tests; Etoposide loaded and folic acid attached PHBHHX nanoparticles were observed as more effective on HeLa cells than Etoposide loaded PHBHHX nanoparticles without attached folic acid. The cytotoxicity of folic acid conjugated PHBHHX nanoparticles to cancer cells was found to be much higher than that of normal fibroblast cells, demonstrating that the folate conjugated nanoparticles has the ability to selectively target to cancer cells. In addition, apoptotic/necrotic activities were evaluated for all formulations of the PHBHHX nanoparticles and parallel results with cytotoxicity tests were obtained. These studies demonstrate that the folic acid attached and Etoposide loaded PHBHHX nanoparticles seem as promising for the targeted cancer therapy.

Bleomycin Loaded Magnetic Chitosan Nanoparticles as Multifunctional Nanocarriers

Iron oxide (Fe3O4) containing magnetic chitosan nanoparticles were prepared with Concanavalin-A and Bleomycin as multifunctional nanocarriers for the targeted cancer therapy by co-precipitation techniques. The chemical structures of nanoparticles were analyzed by FTIR and the magnetic properties of the nanoparticles were evaluated by electron spin resonance technique and vibrational scanning mangnetometer measurements. The in vitro release profiles of Bleomycin were investigated and chitosan nanoparticles characteristics were optimized for subsequent in vivo applications. The magnetic chitosan nanoparticles are biocompatible-based MTT assays. The therapeutic potential of these nanoparticles are being investigated for in vivo applications.