Caner Geyik

Peptide-modified conducting polymer as a biofunctional surface: monitoring of cell adhesion and proliferation

Here, we report the electropolymerization of 3-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)aniline monomer on indium tin oxide (ITO) glass and its use as a coating material for cell culture applications. Functional amino groups on the conducting polymer provide post-modification of the surface with the arginylglycylaspartic acid (RGD) peptide via EDC chemistry. Scanning electron microscopy, atomic force microscopy, and contact angle and surface conductivity measurements were carried out for the surface characterization. The peptide-conjugated surface was tested for adhesion and proliferation of several cell lines such as monkey kidney epithelial (Vero), human neuroblastoma (SH-SY5Y), and human immortalized skin keratinocyte (HaCaT). These cells were cultured on RGD-modified, polymer-coated ITO glass as well as conventional polystyrene surfaces for comparison. The data indicate that the RGD-modified surfaces exhibited better cell adhesion and proliferation among all surfaces compared. Cell imaging studies up to 72 h in length were performed on these surfaces using different microscopy techniques. Therefore, the novel biofunctional substrate is a promising candidate for further studies such as monitoring the effects of drugs and chemicals on cellular viability and morphology as well as cell-culture-on-a-chip applications.

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase-modified gold electrode.

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at −0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation.

Modified gold surfaces by 6-(ferrocenyl)hexanethiol/dendrimer/gold nanoparticles as a platform for the mediated biosensing applications.

An electrochemical biosensor mediated by using 6-(Ferrocenyl) hexanethiol (FcSH) was fabricated by construction of gold nanoparticles (AuNPs) on the surface of polyamidoamine dendrimer (PAMAM) modified gold electrode. Glucose oxidase (GOx) was used as a model enzyme and was immobilized onto the gold surface forming a self assembled monolayer via FcSH and cysteamine. Cyclic voltammetry and amperometry were used for the characterization of electrochemical response towards glucose substrate. Following the optimization of medium pH, enzyme loading, AuNP and FcSH amount, the linear range for the glucose was studied and found as 1.0 to 5.0mM with the detection limit (LOD) of 0.6mM according to S/N=3. Finally, the proposed Au/AuNP/(FcSH+Cyst)/PAMAM/GOx biosensor was successfully applied for the glucose analysis in beverages, and the results were compared with those obtained by HPLC.

Controlled release of anticancer drug Paclitaxel using nano-structured amphiphilic star-hyperbranched block copolymers

In the present study, two amphiphilic star-hyperbranched copolymers based on poly(methyl methacrylate)-b-poly(2-hydroxyethyl methacrylate) (PMMA-b-PHEMA), with different hydrophilic PHEMA segment contents (PMMA-b-PHEMA-1, and PMMA-b-PHEMA-2), were synthesized, and their drug loading and release profiles were examined using Paclitaxel (PTX) as a model drug. The drug loading capacities and encapsulation efficiencies were found to be similar in both polymers. The encapsulation efficiencies were found to be prominent at 98% and 98.5% for PMMA-b-PHEMA-1 and PMMA-b-PHEMA-2, respectively. On the other hand, the drug release behaviors varied in favor of the block copolymer comprising shorter PHEMA chains (PMMA-b-PHEMA-1). Additionally, to assess the biological effects of PTX-loaded polymers, human non-small cell lung carcinoma (A549) cells were used. Cell viability and cell cycle analysis showed that both polymers were non-toxic to cells. The cytotoxic effect of PTX-loaded PMMA-b-PHEMA-1 on A 549 cells was greater (66.49% cell viability at 5.0 ng mL−1 PTX) than that of PMMA-b-PHEMA-2 (72.47% cell viability at 5.0 ng mL−1 PTX), consistent with the drug release experiments.

Offline glucose biomonitoring in yeast culture by polyamidoamine/ cysteamine‐modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at −0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx‐based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R2 = 0.998. Linearity was found to be 0.01–1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R2 = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference‐free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA‐combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.

The covalent bioconjugate of multiwalled carbon nanotube and amino-modified linearized plasmid DNA for gene delivery

Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well‐known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC). The whole pQE‐70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6‐(6‐Amino)hexyl‐2′‐deoxyadenosine‐5′‐triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino‐modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat‐shock step at 42°C. The presence of Ca2+ in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

Complex Structured Fluorescent Polythiophene Graft Copolymer as a Versatile Tool for Imaging, Targeted Delivery of Paclitaxel, and Radiotherapy.

Advances in polymer chemistry resulted in substantial interest to utilize their diverse intrinsic advantages for biomedical research. Especially, studies on drug delivery for tumors have increased to a great extent. In this study, a novel fluorescent graft copolymer has been modified by a drug and targeting moiety and the resulting structure has been characterized by alterations in fluorescent intensity. The polythiophene based hybrid graft copolymer was synthesized by successive organic reactions and combination of in situ N-carboxy anhydride (NCA) ring opening and Suzuki coupling polymerization processes. Initially, targeted delivery of the graft copolymer was investigated by introducing a tumor specific ligand, anti-HER2/neu antibody, on the structure. The functionalized polymer was able to differentially indicate HER2-expressing A549 human lung carcinoma cells, whereas no signal was obtained for Vero, monkey kidney epithelial cells, and HeLa, human cervix adenocarcinoma cells. After integrating paclitaxel into the structure, cell viability, cell cycle progression, and radiosensitivity studies demonstrate HER2/neu targeting polymers were most effective to inhibit cell proliferation. Importantly, the graft copolymer used had no cytotoxic effects to cells, as evidenced by cell viability and cell cycle analysis. This work clearly confirms that a specially designed and fabricated graft copolymer with a highly complex structure is a promising theranostic agent capable of targeting tumor cells for diagnostic and therapeutic purposes.

Poly(p-phenylene) with Poly(ethylene glycol) Chains and Amino Groups as a Functional Platform for Controlled Drug Release and Radiotherapy

Conventional cancer treatments such as chemotherapy, radiotherapy, or combination of these two result in side effects, which lower the quality of life of the patients. To overcome problems with these methods, altering the drug properties by conjugating them to carrier polymers has emerged. Such polymeric carriers also hold the potential to make tumor cells more sensitive to radiation therapy. Herein, poly(p-phenylene) (PPP) polymer with poly(ethylene glycol) (PEG) chains and primary amino groups (PPP-NH2 -g-PEG) is synthesized and conjugated with anticancer drug Doxorubicin (DOX). pH dependent drug release experiments are performed at pH 5.3 and pH 7.4, respectively. Cell viability studies on human cervix adenocarcinoma cells show that lower doses of DOX inhibit cell proliferation when conjugated with nontoxic doses of PPP-NH2 -g-PEG polymer. Additionally, PPP-NH2 -g-PEG/Cys/DOX bioconjugate significantly increases radiosensitive properties of DOX. It is possible to use lower doses of DOX when conjugated to PPP-NH2 -g-PEG in combination with radiotherapy.

Functional poly(p-phenylene)s as targeting and drug carrier materials

ABSTRACT Polymers have a substantial attention in drug delivery systems owing to the diverse intrinsic advantages. It is important to carry the drug to the target site and release to exert its effects. Herein, poly(p-phenylene)s with amino and poly(ethylene glycol) substituents (PPP-NH2-g-PEG) were used as a carrier for doxorubicin (DOX), an anticancer drug, and haloperidol, a sigma receptor targeting ligand. Both human cervix adenocarcinoma cell line (HeLa) and human keratinocyte cell line (HaCaT) having different Sigma receptor 1 (SigmaR1) expression levels were compared. HeLa was found to express twofold SigmaR1 compared to HaCaT cells. Cell imaging studies showed that, DOX cell uptake was higher in HeLa cells when targeted with haloperidol. GRAPHICAL ABSTRACT

pH responsive glycopolymer nanoparticles for targeted delivery of anti-cancer drugs

Over the past decade, there has been a great deal of interest in the integration of nanotechnology and carbohydrates. The advances in glyconanotechnology have allowed the creation of different bioactive glyconanostructures for different types of medical applications, especially for drug delivery and release systems. Therefore, the use of more efficient biocompatible nanocarriers with high loading capacity, low overall toxicity and receptor-mediated endocytosis specificity is still in focus for the enhancement of the therapeutic effect. Conjugation of sugar derivatives onto gold nanoparticles presents unique properties that include a wide array of assembling models and size-related electronic, magnetic and optical properties. Here, pH-responsive drug-conjugated glycopolymer-coated gold nanoparticles were prepared by functionalization of gold nanoparticles with thiol-terminated glycopolymers and then subsequent conjugation of doxorubicin (DOX). Among the four different glycopolymers, their drug release, physicochemical characterization (spectroscopy, particle size and surface charge) and in vitro bioapplications with four different cell lines were compared. As a result, pH-sensitive drug delivery via sugar-coated AuNPs was performed thanks to hydrazone linkages between glycopolymers and DOX. Comparative viability tests also demonstrated the efficiency of glycopolymer–DOX conjugates by fluorescence cell imaging. The obtained results reveal that AuNP homoglycopolymer DOX conjugates (P4D) have significant potential, especially in human neuroblastoma cells in comparison to cervical cancer cells and lung cancer cells.