Mustafa Türk

Intelligent polymers as nonviral vectors

The successful gene therapy largely depends on the vector type that allows a selective and efficient gene delivery to target cells with minimal toxicity. Nonviral vectors are much safer and cheaper, can be produced easily in large quantities, and have higher genetic material carrying capacity. However, they are generally less efficient in delivering DNA and initiating gene expression as compared to viral vectors, particularly when used in vivo. As nonviral vectors, polycations may work well for efficient cell uptake and endosomal escape, because they do form compact and smaller complexes with plasmid DNA and carry amine groups, which give positive charge and buffering ability that allows safe escape from endosome/lysosome. However, this is a disadvantage in the following step, which is releasing the plasmid DNA within the cytosol. In order to initiate transcription and enhance gene expression, the polymer/plasmid complex should dissociate after releasing from endosome safely and effectively. There are also other limitations with some of the polycationic carriers, for example, aggregation, toxicity, etc. Intelligent polymers, also called as ‘stimuli responsive polymers’, have a great potential as nonviral vectors to obtain site-, timing-, and duration period-specific gene expression, which is already exhibited in recent studies that are briefly summarized here.

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.

Gene delivery: intelligent but just at the beginning.

Gene therapy is used to treat genetic disorders, which may be achieved both ex vivo and in vivo. Gene-delivery systems usually include a carrier system which both protects the gene expression plasmid and allows its extracellular and intracellular trafficking. Viruses are used in most of the clinical trials today; however, they do have important drawbacks. Non-viral vectors based on lipids, water-soluble polycations, other non-condensing polymers and nano- or microparticles/capsules have been proposed. Cationic polymers. especially carrying novel targeting ligands. are receiving increasing attention. Intelligent polymers with temperature, pH, and light sensitivities for a controllable and effective non-viral transfection have recently been introduced but are just at the beginning. Our preliminary studies showed that block copolymers of N-isopropylacrylamide-acrylic acid with poly(ethylene imine) could be one example of these novel non-viral vectors.

Biocompatible and biodegradable poly(Tannic Acid) hydrogel with antimicrobial and antioxidant properties

A novel resourceful bulk poly(Tannic Acid) (p(TA)) hydrogel was prepared by crosslinking TA molecules with an epoxy crosslinker, trimethylolpropane triglycidyl ether (TMPGDE), in an autoclave at 90°C for 2h. The obtained p(TA) hydrogels were in disk form and have highly porous morphology. The swelling characteristics of p(TA) hydrogels were investigated in wound healing pH conditions of pH 5.4, 7.4, and 9 at 37.5°C, and the hydrogels showed good swelling and moisture content behavior. Especially, p(TA) hydrogels were found to be sensitive to pH 9 with 1669% maximum swelling. P(TA) hydrogels were completely degraded at pH 9 hydrolytically in 9 days. Total phenol contents and the effects of scavenging ABTS(+) radicals of degraded p(TA) hydrogels at pH 5.4, 7.4, and 9 were evaluated and calculated in terms of gallic acid equivalent and trolox equivalent antioxidant capacity, respectively, and found to be very effective. Moreover, degraded p(TA) hydrogels display strong antimicrobial behavior against gram positive Staphylococcus aureus, Bacillus subtilis, gram negative Pseudomonas aeruginosa bacteria strains and Candida albicans fungus strain. The WST-1 results indicated that bulk p(TA) hydrogels have no cyctotoxicity to the L929 fibroblast cell line in vitro.

Synthesis, characterization and modification of Gum Arabic microgels for hemocompatibility and antimicrobial studies.

Gum Arabic (GA) microgels were successfully prepared via reverse micellization method with high yield (78.5±5.0%) in 5-100μm size range using divinyl sulfone (DVS) as a crosslinker. The GA microgels were degraded hydrolytically 22.8±3.5% at pH 1 in 20days, whereas no degradation was observed at pH 7.4 and pH 9 at 37°C. By using diethylenetriamine (DETA), and taurine (TA) as chemical modifying agents, GA microgels were chemically modified as GA-DETA and GA-TA, and the zeta potential values of 5.2±4.1 and -24.8±1.3mV were measured, respectively in comparison to -27.3±4.2mV for GA. Moreover, blood compatibility of GA, GA-TA, and GA-DETA microgels was tested via in vitro protein adsorption, % hemolysis ratio, and blood clotting index. All the microgels were hemocompatible with% hemolysis ratio between 0.23 to 2.05, and the GA microgels were found to be highly compatible with a blood clotting index of 81±40. The biocompatibility of GA, GA-DETA and GA-Taurine microgels against L929 fibroblast cells also revealed 84.4, 89.1, and 67.0% cell viability, respectively, at 25.0μg/mL concentration, suggesting great potential in vivo biomedical applications up to this concentration. In addition, 5 and 10mg/mL minimum inhibition concentrations of protonated GA-DETA microgels (GA-DETA-HCl) were determined against E. coli and S. aureus, respectively.

Syntheses, structural characterization and biological activities of spiro-ansa-spiro-cyclotriphosphazenes

The replacement reactions of the Cl-atoms in partly substituted spiro-ansa-spiro-cyclotriphosphazenes (7 and 8) with excess pyrrolidine, 4-(2-aminoethyl)morpholine, and 1,4-dioxa-8-azaspiro[4,5]decane in dry THF led to the formation of heterocyclic amine substituted cyclotriphosphazenes (9a–c and 10a–c). All cyclotriphosphazene derivatives were characterized by elemental analysis, FTIR, MS, 1D 1H, 13C and 31P NMR and 2D HSQC, and HMBC techniques, and the crystal structure of partly substituted cyclotriphosphazene 8 was verified by X-ray diffraction analysis. Cyclotriphosphazene derivatives (5–8, 9a–c, and 10a–c) were subjected to antimicrobial activity against seven clinic bacteria and one yeast strain, and the interactions of the phosphazenes with plasmid pBR322 DNA were investigated. Phosphazene derivatives [(5, 7, 8, 9b and 9c) and (10a and 10b)] caused a slight increase and substantial decrease in the mobility of form I DNA, respectively, while 9a caused retardation on gel. Cytotoxic, apoptotic and necrotic effects against L929 fibroblast and A549 lung cancer cells were also evaluated. While the highest toxic effect was obtained for 9a in L929 fibroblast cells and for 9c in A549 lung cancer cells at 100 μg mL−1 concentration, the highest apoptotic effect was determined for 10a in L929 fibroblast cells and for 9a in A549 lung cancer cells at the same concentration. It was found that 9a and 10b exhibited the most necrotic effects against L929 fibroblast and A549 lung cancer cells, respectively. The toxic and necrotic effects of the phosphazenes against A549 lung cancer cells were greater than those against L929 fibroblast cells, whereas, the apoptotic effect of the compounds was greater in L929 fibroblast cells than in A549 lung cancer cells.

Poly(sucrose) micro particles preparation and their use as biomaterials.

Crosslinked p(sucrose) micro particles were synthesized for the first time from sucrose in water-in-oil microemulsion. Using divinyl sulfone (DVS) as crosslinker via reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) p(sucrose) micro particles formed in a single step with very high yield (>90%). The particles have wide size distributions, and negative zeta potential, -27.30 mV, and can be made magnetic field responsive. P(sucrose) particles were shown to be degradable at pHs of 2.5 and 11. Dopamine and gallic acid were used as model drugs for absorption/release studies from p(sucrose) particles. Interestingly, it was shown that p(sucrose) microparticles can be a nutrient for Escherichia coli, and maybe used as a growth medium for other cells, bacteria and organisms. Additionally, the cytotoxic effect of p(sucrose) particles were determined as 26 and 32.5% dead cells against MDA MB-231 cancerous cells and L929 fibroblast cells at 100 ug/ml concentration, respectively. P(sucrose) particles can be safely used for in vivo applications.

Potential c-myc Antisense Oligonucleotide Carriers: PCl/PEG/PEI and PLL/PEG/PEI

Abstract: In this work, positively charged, micelle-forming polymers were synthesized and used as a model vector to deliver antisense oligodeoxynucleotide (ASODN) into melanoma cells. Polymers and polymer/ASODN complexes were characterized by DLS according to size, charge, and critical micelle concentration. Nanosize and spherical complexes were observed by AFM. Complexes did not reveal significant toxicity to melanoma cells. Antiproliferative effect of the complexes was observed by immunocytochemical staining and estimated as 56.8% with N/P:9. High amount of apoptosis and very small amount of necrosis were estimated. According to the results, these positively charged polymers forming micelle-like structures seem promising as ASODN carriers.

Phosphorus–nitrogen compounds. Part 35. Syntheses, spectroscopic and electrochemical properties, and antituberculosis, antimicrobial and cytotoxic activities of mono-ferrocenyl-spirocyclotetraphosphazenes

The reactions of octachlorocyclotetraphosphazene, N4P4Cl8, with N-alkyl-N-mono-ferrocenyldiamines, FcCH2NH(CH2)nNHR1 [n = 2, Fc = ferrocene, R1 = Me (1); n = 2, R1 = Et (2) and n = 3, R1 = Me (3)], led to the formation of monoferrocenyl-spirocyclotetraphosphazenes (4–6). When the reactions were carried out with excess pyrrolidine, morpholine and 1,4-dioxa-8-azaspiro[4,5]decane (DASD), the fully substituted products (4a–6c) were obtained in high yields. The structures of all the phosphazene derivatives were characterized by MS, FTIR, 1H, 13C and 31P NMR, HSQC and HMBC techniques. The crystal structures of 4a and 5a were determined by X-ray crystallography. The electrochemically reversible one-electron oxidation of Fc redox centers was observed for cyclotetraphosphazenes. The fully substituted phosphazenes (4a–6c) were evaluated for their antituberculosis activity against reference strain Mycobacterium tuberculosis H37Rv, and compounds 4a–6a and 5c were found to be active. The antibacterial activities of phosphazenes 4a–6c against G(+) and G(−) bacteria and their antifungal activities against yeast strains were carefully scrutinized. The results indicate that compounds 4a–6a, 6b, 4c and 5c are very effective against yeast strains. The anticandidal activities of 6a and 6b make them promising anticandidal agents. The interactions of these compounds with plasmid DNA and their cytotoxic activity against L929 fibroblast and DLD-1 colon cancer cell lines were also investigated.

Phosphorus–nitrogen compounds. Part 36. Syntheses, Langmuir–Blodgett thin films and biological activities of spiro-bino-spiro trimeric phosphazenes

The condensation reactions of hexachlorocyclotriphosphazene (N3P3Cl6, trimer) with the symmetric N2N2 or N2O2 donor type tetradentate bulky ligands (1–4) gave partly substituted spiro-bino-spiro (sbs) (5–8) trimeric phosphazenes. Compounds 5–8 reacted with pyrrolidine, morpholine and 1,4-dioxa-8-azaspiro[4,5]decane (DASD) to give octapyrrolidino- (9–12), morpholino- (13–16) and DASD-substituted cyclotriphosphazenes (17–20). The structures of the phosphazenes have been elucidated using FTIR, MS, 1H, 13C{1H} and 31P{1H} NMR, and HSQC spectral data. The molecular and solid-state structures of 5, 6 and 12 were verified by single crystal X-ray diffraction techniques. On the other hand, the ultrathin and highly ordered Langmuir–Blodgett (LB) films of compounds 6, 7, 9 and 12 were also fabricated. The structural characterization of the LB films was made using p-polarized grazing angle (GAIR) and horizontal attenuated total reflectance (HATR) techniques. All the novel phosphazene derivatives were evaluated for antibacterial activities against Gram-positive (G+) and Gram-negative (G−) bacteria and for antifungal activities against yeast strains. In addition, the cytotoxic effects of compounds 9, 13, 15, 16, 19 and 20 were investigated against L929 fibroblast and MDA-MB-231 breast cancer cells. The most active one among these compounds was compound 9 at 6.25 μg mL−1 concentration. The interactions between compounds 5–20 and pBR322 plasmid DNA were determined by agarose gel electrophoresis.