Nanosized multifunctional liposomes for tumor diagnosis and molecular imaging by SPECT/CT.

Among currently used cancer imaging methods, nuclear medicine modalities provide metabolic information, whereas modalities in radiology provide anatomical information. However, different modalities, having different acquisition times in separate machines, decrease the specificity and accuracy of images. To solve this problem, hybrid imaging modalities were developed as a new era, especially in the cancer imaging field. With widespread usage of hybrid imaging modalities, specific contrast agents are essentially needed to use in both modalities, such as single-photon emission computed tomography/computed tomography (SPECT/CT). Liposomes are one of the most desirable drug delivery systems, depending on their suitable properties. The aim of this study was to develop a liposomal contrast agent for the diagnosis and molecular imaging of tumor by SPECT/CT. Liposomes were prepared nanosized, coated with polyethylene glycol to obtain long blood circulation, and modified with monoclonal antibody 2C5 for specific tumor targeting. Although DTPA-PE and DTPA-PLL-NGPE (polychelating amphilic polymers; PAPs) were loaded onto liposomes for stable radiolabeling for SPECT imaging, iopromide was encapsulated into liposomes for CT imaging. Liposomes [(DPPC:PEG(2000)-PE:Chol:DTPA-PE), (PL 90G:PEG(2000)-PE:Chol:DTPA-PE), (DPPC:PEG(2000)-PE:Chol:PAPs), (PL 90G:PEG(2000)-PE:Chol:PAPs), (60:0.9:39:0.1% mol ratio)] were characterized in terms of entrapment efficiency, particle size, physical stability, and release kinetics. Additionally, in vitro cell-binding studies were carried out on two tumor cell lines (MCF-7 and EL 4) by counting radioactivity. Tumor-specific antibody-modified liposomes were found to be effective multimodal contrast agents by designating almost 3-8 fold more uptake than nonmodified ones in different tumor cell lines. These results could be considered as an important step in the development of tumor-targeted SPECT/CT contrast agents for cancer imaging.

Evaluation of the cytotoxicity of different root canal sealers on L929 cell line by MTT assay.

The purpose of this study was to evaluate the cytotoxic effects of commercially available root canal sealers [Sealite Ultra (SU), Tubli-Seal (TS), Tubli-Seal EWT (TS-EWT), Pulp Canal Sealer (PCS), Pulp Canal Sealer EWT (PCS-EWT), Endomethasone N (En N), and Apexit Plus (AP)] on L929 cells by using MTT assay. After incubation with each sealer's extract at 37°C in a humidified air atmosphere containing 5% CO(2 )for 24 h, MTT (5 mg/mL) in saline was added into each well and further incubated at 37°C for 4 h. Formazan precipitate was dissolved in a buffer containing 23% sodium dodecyl sulfate and 50% N, N-dimethylformamide (pH 4.7). Optical densities of dissolved formazan were read using a microplate spectrophotometer. AP, TS, and TS-EWT showed no cytotoxicity at any dilution tested. Other sealers exhibited some degree of cytotoxicity at the 1/4 and 1/2 dilutions. PCS-EWT and SU exerted more potent cytotoxicity at 1/2 dilution than the other sealers.

Preparation and in vitro evaluation of meloxicam-loaded PLGA nanoparticles on HT-29 human colon adenocarcinoma cells.

CONTEXT: The inhibitors of cyclooxygenase (COX)-2 play an important role in cancer chemoprevention. Certain COX-2 inhibitors exert antiproliferative and pro-apoptotic effects on cancer cells. OBJECTIVE: In this study, meloxicam, which is an enolic acid-type preferential COX-2 inhibitor, was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to maintain local high concentration, and its efficacy was determined. METHODS: NPs were prepared by using salting-out and emulsion-evaporation steps. Meloxicam-loaded NP formulations were evaluated with respect to the drug loading, particle size, polydispersity index, zeta potential, drug release rate, and residual poly(vinyl alcohol) (PVA) percentage. The effects of PLGA and PVA molecular weight variations on the physicochemical properties of NPs were investigated. Stability of meloxicam in NPs was assessed over 3 months. COX-2 expressing human colon adenocarcinoma cell line HT-29 was used in cellular uptake and viability assays. RESULTS: NPs had a spherical shape and a negative zeta potential, and their size ranged between 170-231 nm with a lower polydispersity index. NPs prepared with high molecular weight PLGA were shown to be physically stable over three months at 4°C. The increase in molecular weight of the polymer and emulsifier reduced the in vitro release rate of meloxicam from NPs. Meloxicam-loaded NPs showed cytotoxic effects on HT-29 cells markedly at 800 µM. Cancer cells had high uptake of coumarin-6-loaded NPs. CONCLUSION: The PLGA NPs developed in this study can be a potentially effective drug delivery system of meloxicam for the treatment of colon cancer.

Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors.

Cationic nanoparticles of chitosan (CS), poly-epsilon-caprolactone coated with chitosan (CS-PCL) and poly-epsilon-caprolactone coated with poly-L-lysine (PLL-PCL) were developed to encapsulate intravesical chemotherapeutic agent Mitomycin C (MMC) for longer residence time, higher local drug concentration and prevention of drug loss during bladder discharge. Nanoparticle diameters varied between 180 and 340 nm depending on polymer used for preparation and coating. Zeta potential values demonstrated positive charge expected from cationic nanoparticles. MMC encapsulation efficiency depended on hydrophilicity of polymers since MMC is water-soluble. Encapsulation was increased by 2-fold for CS-PCL and 3-fold for PLL-PCL as a consequence of hydrophilic coating. Complete drug release was obtained with only CS-PCL nanoparticles. On the other hand, CS and PLL-PCL nanoparticles did not completely liberate MMC due to strong polymer-drug interactions which were elucidated with DSC studies. As far as cellular interaction was concerned, CS-PCL was the most efficient formulation for uptake of fluorescent markers Nile Red and Rhodamine123 incorporated into nanoparticles. Especially, CS-PCL nanoparticles loaded with Rhodamine123 sharing hydrophilic properties with MMC were selectively incorporated by bladder cancer cell line, but not by normal bladder epithelial cells. CS-PCL nanoparticles seem to be promising for MMC delivery with respect to anticancer efficacy tested against MB49 bladder carcinoma cell line.

Safety and efficacy of amphiphilic beta-cyclodextrin nanoparticles for paclitaxel delivery.

Paclitaxel is a potent anticancer agent with limited bioavailability due to side-effects associated with solubilizer used in its commercial formulation and the tendency of the drug to precipitate in aqueous media. In this study, paclitaxel was encapsulated in amphiphilic cyclodextrin nanoparticles. Safety of blank nanoparticles was compared against commercial vehicle cremophor:ethanol (50:50 v/v) by hemolysis and cytotoxicity experiments. Data revealed that nanoparticles caused significantly less hemolysis. Results were confirmed with SEM imaging of erythrocytes treated with nanospheres, nanocapsules or commercial vehicle. Cytotoxicity of the blank carriers was evaluated against L929 cells. A vast difference between the cytotoxicity of nanoparticles and cremophor:ethanol mixture was observed. Physical stability of paclitaxel in nanoparticles was assessed for 1 month with repeated particle size and zeta potential measurements and AFM imaging. Recrystallization of paclitaxel, very typical in diluted aqueous solutions of the drug, did not take place when the drug is bound to cyclodextrin nanoparticles. Anticancer efficacy of paclitaxel-loaded nanoparticles was evaluated in comparison to paclitaxel in cremophor vehicle against MCF-7 cells. Cyclodextrin nanoparticle caused a slightly higher anticancer effect than cremophor:ethanol vehicle. Thus, amphiphilic cyclodextrin nanoparticles emerged as promising alternative formulations for injectable paclitaxel administration with low toxicity and equivalent efficacy.

The comparative effects of gene modulators on thyroid-specific genes and radioiodine uptake.

The aim of this study was to comparatively investigate the effects of 5-azacytidine-C (5-Aza), trichostatin-A (TSA), and all-trans retinoic acid (ATRA) on the mRNA expressions of the sodium and iodine (Na/I) symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyroid-stimulating hormone receptor (TSH-R), as well as radioiodine (RAI) uptake in cancer (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines. Cell lines were treated with 10 ng/mL of TSA, 5 microM of 5-AZA, and 1 microM of ATRA, according to the MTT (methyl-thiazol-tetrazolium) test results. Additionally, recombinant thyroid-stimulating hormone (rTSH) was also applied, with a selected dose of 100 ng/mL. Following the treatment, NIS, Tg, TPO, and TSH-R mRNA levels were detected by real-time-polymerase chain reaction (RT-PCR) and RAI uptakes were measured by using a well counter as counts/cell number. 5-Aza increased TSH-R mRNA expression in both of the cell lines and decreased TPO, NIS, and Tg mRNA levels in the cancer cell line. In the normal thyroid cell line, 5-AZA increased TPO mRNA levels by 2-fold and made no differences in NIS and Tg mRNA levels. TSA treatment repressed NIS and Tg mRNA levels and made no change on other thyroid-specific genes that were investigated in the cancer cell line. In the normal thyroid cell line, TSA increased TSH-R mRNA levels in 72 hours and created no important difference in the other genes. ATRA repressed the TSH-R mRNA levels in the normal thyroid cell line and increased the TPO and Tg mRNA levels slightly in both the cell lines. Furthermore, in short-term treatment, ATRA repressed the NIS gene expression slightly, but in the long term, this repression turned to basal levels. 5-Aza, TSA, and ATRA did not make any changes in RAI uptake in the cancer cell line, but rTSH increased RAI uptake significantly. In the normal thyroid cell line, TSA and ATRA decreased RAI uptake (to 1/10 and 1/2, respectively), but 5-Aza and rTSH increased RAI uptake significantly (2- and 4-fold, respectively). In our study, we showed an increase in TSH-R gene expression and radioiodine uptake with 5-Aza. Further in vitro and in vivo studies are needed to support our findings and the potential clinical use of this agent.

The comparative effects of gene modulators on thyroid-specific genes and radioiodine uptake.

The aim of this study was to comparatively investigate the effects of 5-azacytidine-C (5-Aza), trichostatin-A (TSA), and all-trans retinoic acid (ATRA) on mRNA expressions of Na/I symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), and thyroid stimulating hormone receptor (TSH-R), and radioiodine (RAI) uptake in cancer (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines. Cell lines were treated with 10 ng/mL of TSA, 5 microM of 5-Aza, and 1 microM of ATRA, according to the MTT (methyl-thiazol-tetrazolium) test results. Additionally, recombinant thyroid stimulating hormone (rTSH) was also applied, with a selected dose of 100 ng/mL. Following the treatment, NIS, Tg, TPO, and TSH-R mRNA levels were detected by real-time-polymerase chain reaction (RT-PCR) and RAI uptakes were measured by using a well counter as the counts/cell number. 5-Aza increased TSH-R mRNA expression in both of the cell lines and decreased TPO, NIS, and Tg mRNA levels in the cancer cell line. In the normal thyroid cell line, 5-Aza increased TPO mRNA levels 2-fold and made no differences in NIS and Tg mRNA levels. TSA treatment repressed NIS and Tg mRNA levels, and made no differences on other thyroid specific genes investigated in the cancer cell line. In the normal thyroid cell line, TSA increased TSH-R mRNA levels in 72 hours and created no important differences in other genes. ATRA repressed the TSH-R mRNA levels in the normal thyroid cell line and increased the TPO and Tg mRNA levels slightly in both cell lines. Furthermore, in short-term treatment, ATRA repressed NIS gene expression slightly, but in the long term, this repression turned to basal levels. 5-Aza, TSA, and ATRA did not make any differences in RAI uptake in the cancer cell line, but rTSH increased RAI uptake significantly. In the normal thyroid cell line, TSA and ATRA decreased RAI uptake (to 1/10 and 1/2, respectively), but 5-Aza and rTSH increased RAI uptake significantly (2- and 4-fold, respectively). We have shown an increase in TSH-R gene expression and radioiodine uptake with 5-Aza. Further in vitro and in vivo studies are needed to support our findings and the potential clinical use of this agent.

Water soluble distyryl-boradiazaindacenes as efficient photosensitizers for photodynamic therapy.

We introduce a novel class of water soluble, extended conjugation boradiazaindacene dyes which are efficient singlet oxygen generators and have spectacular photoinduced cytotoxicity when excited in the "therapeutic window" of the electromagnetic spectrum.

Cytotoxic evaluation of injectable cyclodextrin nanoparticles.

Nanoparticles were prepared using beta-CDC6, which is an amphiphilic beta-cyclodextrin derivative modified on the secondary face with 6C aliphatic esters. A nanoprecipitation technique was used to prepare the blank nanoparticles without any surfactant and nanoparticles containing Pluronic F68 as surfactant in a concentration range of 0.1 to 1%. Nanoparticle formulations were characterized by particle size distribution and zeta potential measurements. Entrapment efficiency and in-vitro release profiles were determined and the cytotoxicity of these injectable nanospheres was evaluated against mouse fibroblast L929 cell line and human polymorphonuclear cells by methlythiazolyltetrazolium assay. As far as particle size and zeta potential are concerned, there is a relationship between surfactant presence and nanoparticle characteristics. However, these effects are not significant. It was also found that surfactant presence has no effect on model drug nimodipine encapsulation but accelerates the in-vitro release of the drug. Cell culture studies on mouse fibroblasts and human polymorphonuclear cells revealed a concentration-dependent cytotoxicity more pronounced in fibroblast cells. This led to the conclusion that the use of surfactants in injectable nanoparticles prepared from amphiphilic beta-cyclodextrins may lead to altered in-vitro properties and impaired safety for the drug delivery system.

Water-soluble green perylenediimide (PDI) dyes as potential sensitizers for photodynamic therapy.

[reaction: see text] A series of water-soluble green perylenediimide (PDI) dyes have been synthesized. On red light excitation, these dyes were shown to be efficient generators of singlet oxygen, and in cell culture media, they were shown to display significant light-induced cytotoxic effects on the human erythroleukemia cell line (K-562). It appears that highly versatile PDI dyes are likely to find new applications in photodynamic therapy.

Effect of fludarabine on leukocyte functions.

BACKGROUND: Fludarabine induces leukemic cell apoptosis and is highly efficient in chronic lymphocytic leukemia. However, fludarabine therapy causes severe leukopenia. Leukocyte myeloperoxidase (MPO) catalyzes the formation of HOCl, and this is the main microbicidal function in phagocytes. The aim of our study was to evaluate the effect of fludarabine on leukocytes, i.e. their degranulation capacity, MPO activity and HOCl production. METHODS: Peripheral blood leukocytes were incubated for 48 h with fludarabine. Degranulation was measured using a flow-cytometric method. MPO activity and HOCl production were measured spectrophotometrically. RESULTS: The degranulation capacity of fludarabine-treated leukocytes was significantly elevated compared to untreated controls. MPO activity and HOCl production were also increased in parallel. A possible direct activating effect of fludarabine was tested on the MPO activity of HL60 cells. Fludarabine did not affect MPO activity at concentrations ranging from 10 microM to 2 mM. CONCLUSION: Fludarabine had no inhibitory effect on the microbial killing of leukocytes.

Evaluation and comparison of MRP1 activity with three fluorescent dyes and three modulators in leukemic cell lines.

MRP1 activity was evaluated and compared in 11 cell lines with different levels of MRP1 expression using functional assays of calcein acetoxymethyl ester (calcein-AM), carboxyfluorescein diacetate (CFDA) and Rhodamine 123 (Rh123) in combination with the modulators cyclosporin A (CsA), probenecid and MK571. A good correlation was found between MRP1 expression and the modulatory effect of MK571 on calcein-AM uptake (P = 0.01 and probenecid effect on CFDA uptake (P = 0.02). Additionally, the combined modulatory effect of MK571 and probenecid on CFDA uptake (P < 0.0001) and on calcein-AM uptake (P = 0.0001) were highly significant. No correlation was found between MRP1 expression and the effects of three modulators on Rh123 uptake or efflux. In conclusion, calcein-AM and CFDA uptake assays are the best choices to probe MRP1 activity and combination of two modulators may improve the efficiency of these assays.