Murat Demirbilek

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.

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.

Evaluation of cytotoxic and mutagenic effects of Coriolus versicolor and Funalia trogii extracts on mammalian cells.

This study examined the in vitro cytotoxic activities of standardized aqueous bioactive extracts prepared from Coriolus versicolor and Funalia trogiiATCC 200800 on HeLa and fibroblast cell lines using a MTT (3-[4,5-dimetiltiazol-2-]-2–5-difeniltetrazolium bromide) cytotoxicity assay. F. trogii and C. versicolor extracts were cytotoxic to both cell lines. At 10 μL treatment level, F. trogii and C. versicolor extracts inhibited proliferation of HeLa cancer cells by 71.5% and 45%, respectively, compared with controls. Toxicity was lower toward normal fibroblasts. In the latter case, treatment at 10 μL level with F. trogii and C. versicolor extracts reduced cell proliferation by 51.3% and 38.7%, respectively. In separate experiments, the mitotic index (MI) obtained with 3 μL treatment level of unheated extracts of the two fungi was comparable to the MI value obtained by treatment with 4 μg/mL MMC (anticancer agent mitomycin-C). A significant induction of sister chromatid exchange (SCE) was observed in normal cultured lymphocytes treated with MMC (4 μg/mL). MMC treatment reduced replication index compared with treatment with unheated F. trogii extract and negative controls (p < 0.001). In contrast to MMC, F. trogii extracts did not affect the proliferation of human lymphocytes compared with controls (p > 0.05). Laccase and peroxidase enzyme activities in F. trogii extract were implicated in their inhibitory effect on cancer cells. F. trogii extract was concluded to have antitumor activity.

The effect of poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) (PHBHHx) and human mesenchymal stem cell (hMSC) on axonal regeneration in experimental sciatic nerve damage

This study is designed to evaluate the treatment effect of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and human mesenchymal stem cells (hMSC) on axonal regeneration in experimental rat sciatic nerve damage, and compare the results of this modality with autologous nerve grafting. In Spraque–Dawley albino rats, 10-mm-long experimental nerve gaps were created. Three groups were constituted, the gap was repaired with autologous nerve graft (autograft group), PHBHHx nerve graft alone (PHBHHx alone group), and PHBHHx nerve graft with hMSCs inside (PHBHHx with hMSC group), respectively. The results were evaluated with functional recovery, electrophysiological evaluation, and histological evaluation either with light microscopy and transmission electron microscopy for axonal regeneration and myelin formation. In functional evaluation, autograft and PHBHHx with hMSC groups showed functional improvement with time, whereas PHBHHx alone group did not. Electrophysiological evaluation showed better results in autograft and PHBHHx with hMSC groups when compared to PHBHHx alone group. There was no statistical difference between autograft and PHBHHx with hMSC groups. Histological evaluation showed regenerated axons in each group. Autograft group was better than the others, and PHBHHx with hMSC group was better than PHBHHx alone group both for axonal regeneration and myelin formation. This study showed that the nerve grafts which were prepared from PHBHHx with oriented nanofiber three-dimensional surfaces aided to nerve regeneration, either used alone or with hMSC. PHBHHx provided better nerve regeneration when used with hMSCs inside than alone, and reached the same statistical treatment effect in functional evaluation and electrophysiological evaluation when compared to autografting.

Comparative evaluation of cyclosporine A/HPβCD-incorporated PLGA nanoparticles for development of effective ocular preparations

To improve poor water solubility of cyclosporine A (CsA), hydroxypropyl-beta-cyclodextrin (HPβCD) was incorporated into the nanoparticle formulation. Solid complexes of CsA with HPβCD in different ratios were prepared by the kneading method. CsA containing alone or in combination with HPβCD in poly-lactide-co-glycolide (P-CsA or P-CsA-HPβCD) nanoparticles were prepared by the emulsification solvent evaporation method. The mean size of CsA-loaded NPs was found to be approximately 220 nm. The solubility of CsA was significantly improved and the phase solubility diagram of CsA–HPβCD systems showed an AL type phase. Nanoparticles showed high CsA encapsulation efficiency (88%) and production yield (89%). Release rate was increased by the presence of HPβCD and total cumulative release ranged from 75% to 96% in 24 h. In vitro cytotoxicity study assay resulted in a low toxicity for all types of nanoparticles. After 6 h incubation period, the cellular uptake was found at 33% and 32% for P–CsA and P–HPβCD–CsA nanoparticles, respectively.

Chlorogenic Acid Containing Bioinspired Polyurethanes: Biodegradable Medical Adhesive Materials

Highly adhesive bioinspired polyurethanes (PUs) based on the chlorogenic acid (CLA) were prepared from 4,4′-methylenebis(cyclohexyl isocyanate) (MCI) and polyethylene glycol 200 (PEG 200). The polyurethanes exhibited the lowest glass transition temperature (Tg), improved thermal stability and good adhesive properties. The highest adhesion strength was found as 373.3 ± 47.5 kPa for 10% CLA containing PUs. The polyurethanes were observed to be biodegradable and were in the range of 19–24% for 8 weeks as depending on chlorogenic acid containing of PUs. As a result, prepared biocompatible-adhesive bioinspired polyurethanes are good candidates for medical applications as a tissue adhesive material. GRAPHICAL ABSTRACT

Magnetic silk fibroin e-gel scaffolds for bone tissue engineering applications:

Recently, the incorporation of magnetic nanoparticles into standard scaffolds has emerged as a promising approach for tissue engineering applications. This strategy can promote not only tissue regeneration but also reloading of scaffolds through an external supervising center that adsorbs growth factors, preserving their stability and biological activity. In this study, novel magnetic silk fibroin e-gel scaffolds were prepared by the electrogelation process of concentrated Bombyx mori silk fibroin (8 wt%) aqueous solution. In addition, basic fibroblast growth factor was conjugated physically to human serum albumin = Fe3O4 nanoparticles (71.52 ± 2.3 nm in size) with 97.5% binding yield. Scanning electron microscopy images of the prepared human serum albumin = Fe3O4-basic fibroblast growth factor-loaded silk fibroin e-gel scaffolds showed a three-dimensional porous morphology. In terms of water uptake, basic fibroblast growth factor-conjugated scaffolds had the highest water absorbability among all groups. In vitro cell culture studies showed that both the human serum albumin coating of Fe3O4 nanoparticle surface and basic fibroblast growth factor conjugation had an inductive effect on cell viability. One of the most used markers of bone formation and osteoblast differentiation is alkaline phosphatase activity; human serum albumin = Fe3O4-basic fibroblast growth factor-loaded silk fibroin e-gels showed significantly enhanced alkaline phosphatase activity (p < 0.05). SaOS-2 cells cultured on human serum albumin = Fe3O4-basic fibroblast growth factor-loaded silk fibroin e-gels deposited more calcium compared with those cultured on bare silk fibroin e-gels. These results indicated that the proposed e-gel scaffolds are valuable candidates for magnetic guiding in bone tissue regeneration, and they will present new perspectives for magnetic field application in regenerative medicine.

Surface-modified bacterial nanofibrillar PHB scaffolds for bladder tissue repair

The aim of the study is in vitro investigation of the feasibility of surface-modified bacterial nanofibrous poly [(R)-3-hydroxybutyrate] (PHB) graft for bladder reconstruction. In this study, the surface of electrospun bacterial PHB was modified with PEG- or EDA via radio frequency glow discharge method. After plasma modification, contact angle of EDA-modified PHB scaffolds decreased from 110 ± 1.50 to 23 ± 0.5 degree. Interestingly, less calcium oxalate stone deposition was observed on modified PHB scaffolds compared to that of non-modified group. Results of this study show that surface-modified scaffolds not only inhibited calcium oxalate growth but also enhanced the uroepithelial cell viability and proliferation.

Aligned bacterial PHBV nanofibrous conduit for peripheral nerve regeneration

Abstract The conventional method of peripheral nerve gap treatment is autografting. This method is limited. In this study, an aligned nanofibrous graft was formed using microbial polyester, Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The regenerative effect of the graft was compared with that of autografting in vivo. To determine the regenerative effect, rats were assessed with sciatic nerve functional index, electromyographic evaluation, and histological evaluation. Results found in this study include PHBV grafts stimulated progressive nerve regeneration, although regeneration was not comparable with that of autografting. We conclude that the study results were promising for aligned bacterial polymeric grafts for peripheral nerve regeneration.