Fibroblast Cell Line L929 Research Articles

Removal of cadmium from aqueous solutions by oxidized and ethylenediamine-functionalized multi-walled carbon nanotubes

Surface functionalization of multi-walled carbon nanotubes (MWCNTs) by ethylenediamine, via chemical modification of carboxyl groups, using O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, was performed. The resulting materials were characterized by different techniques, such as FTIR, TGA and elemental analysis. Biocompatibility studies showed that the functionalized MWCNTs, at concentrations between 1 and 50 μg mL−1, were not cytotoxic for the fibroblast L929 cell line. In batch tests, the influences of solution pH, contact time, initial metal ion concentration and temperature on the sorption of Cd2+ ions onto raw-MWCNTs (raw-MWCNT), oxidized MWCNTs (o-MWCNT) and ethylenediamine-functionalized MWCNTs (e-MWCNT) were studied. The adsorption of Cd2+ ions by o-MWCNT and e-MWCNT was strongly pH dependent. The time dependent Cd2+ sorption onto raw-MWCNT, o-MWCNT and e-MWCNT can be described by a pseudo-second-order kinetic model. The Langmuir isotherm model agrees well with the equilibrium experimental data. The maximum capacity was obtained for e-MWCNT, 25.7 mg g−1, at 45 °C. The thermodynamic parameters were also deduced for the adsorption of Cd2+ ions on raw-MWCNT, o-MWCNT and e-MWCNT and the results showed that the adsorption was spontaneous and endothermic.

A new approach for the in vitro identification of the cytotoxicity of superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are increasingly used in medical applications, such as targeting delivery and imaging. In the future, patients are more likely to be exposed to pharmaceutical products containing such particles. The study of toxicity of SPIONs has become of great importance in recent years, although the published data in this arena is limited. The aim of the present work is to investigate the cytotoxicity of SPIONs and the effect of the particles on the cell medium components. For this purpose, uncoated and polyvinyl alcohol (PVA) coated SPIONs with narrow size distribution were synthesized via a well-known coprecipitation method. The mouse fibroblast cell line L929 was exposed to SPIONs to probe the toxicity of magnetic nanoparticles during the bio application. Changes to the cell medium caused by SPIONs were analyzed with zeta potential measurements, ultraviolet visible spectroscopy (UV/vis) and the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. It is observed that gas vesicles are formed in SPION-treated cells. Toxicity is conventionally explained by changes in the DMEM's pH and composition due to the tendency of SPIONs to interact with biomolecules. A new procedure is proposed to examine the in vitro toxicity of nanoparticles in a more rigorous manner, which gives an improvement in the relationship between in vivo and in vitro toxicity studies.

A simple method to measure cell viability in proliferation and cytotoxicity assays

Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 etam and resorufin at 570 etam wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 etam) and green (500 to 600 etam) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r(2) = 0.996; p < 0.01) and with the cellular concentrations (r(2) = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

Cell adhesion and tissue response to hydroxyapatite nanocrystal-coated poly(l-lactic acid) fabric

Cell adhesion and tissue response to poly(l-lactic acid) (PLLA) fabric coated with nanosized hydroxyapatite (HAp) crystals were studied. The HAp nanocrystals were prepared by the wet chemical process followed by calcination at 800 degrees C with an anti-sintering agent to prevent calcination-induced sintering. After the PLLA fabric was hydrolyzed with an alkaline aqueous solution, the HAp nanocrystals were coated via ionic interaction between the calcium ions on the HAp and the carboxyl groups on the alkali-treated PLLA. The PLLA surface uniformly coated with the HAp nanocrystals was observed by scanning electron microscope. The ionic interaction between the HAp and the PLLA was estimated by FT-IR. Improved cell adhesion to the HAp nanocrystal-coated surface was demonstrated by in vitro testing using a mouse fibroblast cell line L929. Furthermore, reduced inflammatory response to the HAp nanocrystal-coated PLLA fabric (as compared with a non-treated one) was confirmed by a subcutaneous implantation test with rats. Thus the HAp nanocrystal-coated PLLA developed has possible efficacy as an implant material in the fields of general and orthopedic surgery, and as a cell scaffold in tissue engineering.

Supercooled smectic nanoparticles: Influence of the matrix composition and in vitro cytotoxicity

Cholesteryl nonanoate (CN), myristate (CM), palmitate (CP) and oleate (CO) alone or in combination were evaluated as matrix lipids for the preparation of supercooled smectic nanoparticles with a high stability against recrystallization during storage. The phase behavior of the cholesterol esters in the bulk was studied by polarizing light microscopy, differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). Colloidal dispersions with pure and mixed cholesterol ester matrices were prepared by high-pressure melt homogenization and characterized by photon correlation spectroscopy, laser diffraction combined with polarizing intensity differential scattering, DSC and SAXS. The morphology of selected formulations was studied by freeze-fracture electron microscopy. All smectic nanoparticles with a mixed cholesterol ester matrix were stable against recrystallization when stored at room temperature. Nanoparticles with a pure CN and mixed CM/CN matrix with a high fraction of CN (60% of the whole lipid matrix) could even be stored at 4 °C for at least 18 months without any recrystallization. As smectic nanoparticles are studied especially with regard to parenteral administration of lipophilic drugs, the cytotoxicity of selected formulations was compared with that of a clinically used colloidal fat emulsion (Lipofundin MCT) in the murine fibroblast cell line L929 using the sulforhodamine B assay. The supercooled smectic nanoparticle formulations display a good overall cell compatibility although in some cases their cytotoxicity was slightly higher than that of Lipofundin MCT.

Mammalian Reoviruses: Propagation, Quantification, and Storage

Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. This unit describes techniques for culturing mouse fibroblast L929 cell lines, the preferred cell line in which most mammalian reovirus studies take place. In addition, mammalian reovirus propagation, quantification, purification, and storage are described.

Anti‐Inflammatory, Antiproliferative, and Radical‐Scavenging Activities of Tolfenamic Acid and Its Metal Complexes

Some new complexes of tolfenamic acid (=2-[(2-methyl-3-chlorophenyl)amino]benzoic acid; Htolf) with potentially interesting biological activities are described. The complexes [Mn(tolf)(2)(H(2)O)(2)], [Co(tolf)(2)(H(2)O)(2)], [Ni(tolf(2)(H(2)O)(2)], [Cu(tolf)(2)(H(2)O)](2), and [Zn(tolf)(2)(H(2)O)] were prepared by the reaction of tolfenamic acid, a potent anti-inflammatory drug, with metal salts. The radical-scavenging activities of the complexes were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay. Their ability to inhibit soybean lipoxygenase, beta-glucuronidase, and trypsin-induced proteolysis was studied. Their inhibitory effects on rat paw edema induced by carrageenin was studied and compared with those of tolfenamic acid. The complex [Zn(tolf)(2)(H(2)O)] exhibited the strongest in vivo inhibitory effect at 0.1 mm/kg Body Weight (BW; 93.0+/-0.9%), superior than the inhibition induced by tolfenamic acid at the same molar dose (76.0+/-0.9%). Tolfenamic acid and its metal complexes have been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines, MCF-7 (breast cancer cell line), T24 (bladder cancer cell line), and A-549 (non-small cell lung carcinoma), and a mouse fibroblast L-929 cell line. The complexes [Mn(tolf)(2)(H(2)O)(2)] and [Cu(tolf)(2)(H(2)O)](2) have shown selectivity against T24 cell line. The IC(50) values of these two complexes against T24 cancer cell lines are in a micromolar range similar or better to that of the antitumor drug cisplatin.

Synthesis, characterization and cytotoxicity of surface amino-functionalized water-dispersible multi-walled carbon nanotubes

Surface functionalization of multi-walled carbon nanotubes (MWCNTs), with amino groups via chemical modification of carboxyl groups introduced on the nanotube surface, using O-(7-azabenzotriazol-1-yl)- N, N, N′, N′-tetramethyluronium hexafluorophosphate (N-HATU) and N, N-diisopropylethylamine (DIEA) is reported. The N-HATU coupling agent provides faster reaction rate and the reaction occurs at lower temperature compared to amidation and acylation–amidation chemistry. The amines, 1,6-hexanediamine (HDA), diethylenetriamine (DETA), triethylenetetramine (TETA) and 1,4-phenylenediamine (PDA) were used. The resulting materials were characterized with different techniques such as FTIR, XRD, elemental analysis, TGA, TEM, UV–vis spectroscopy and cyclic voltammetry. MWCNTs functionalized with PDA posses the best dispersibility and electron transfer properties in comparison to the others amines. Functionalized MWCNTs, at the concentrations between 1 and 50 μg ml −1, were not cytotoxic for the fibroblast L929 cell line. However, the concentrations of MWCNTs higher of 10 μg ml −1 reduced cell growth and this effect correlated positively with the degree of their uptake by L929 cells.

Pharmakafreisetzende biodegradierbare Polymerbeschichtung von dentalen Titanimplantaten zur Verbesserung der Weichgewebsintegration / Drug release of coated dental implant neck region to improve tissue integration

In order to improve tissue integration, the neck region of dental implants was coated with the biodegradable polymer poly (L-lactide) incorporating tetracycline, ibuprofen and the combination of both drugs using a solvent dip-coating process. Metallographic analysis, light microscopy and electron microscopy were used to detect the thickness range and the surface characteristics of the coatings. Cytotoxicity was evaluated using the tetrazolium colorimetric method with the fibroblast cell line L929. The in vitro drug release was measured in isotonic sodium chloride solution by UV spectroscopy. To explore if drug release is concentration-dependent, the total amount of drug was varied in the coating (20% wt, 30% wt and 40% wt). The results showed a continuous release of the embedded drugs in relevant dosage over a period of 6 months. In contrast to high tetracycline concentrations, high ibuprofen concentrations resulted in a decreased metabolic activity of the L929 fibroblasts.

Housekeeping gene expression is affected by antidepressant treatment in a mouse fibroblast cell line

Quantitative real-time polymerase chain reaction (PCR) is an effective approach in investigating the effects of exogenous compounds on gene expression. This is often achieved by exploiting so-called 'housekeeping' genes as baseline controls to normalise expression levels, which have historically been assumed to have a relatively stable expression pattern. Recent non-in-vitro studies have questioned the validity of this, but previous in-vitro data were lacking following antidepressant treatment. We here investigated the stability of 12 housekeeping genes during treatment of the mouse L929 fibroblast cell line with escitalopram and nortriptyline. Cells were cultured in the presence of antidepressant at 1 microM or 10 microM for 30 min, 24 h or 48 h, and RNA subjected to quantitative PCR (qPCR). Stability of relative transcript expression values was assessed via gene-gene expression ratios and intra- and inter-group variation (using geNorm and NormFinder programs). The three most stable transcripts were adenosine triphosphate (ATP) synthase, H+ transporting mitochondrial F1 complex, beta subunit, beta-2 microglobulin and cytochrome c-1. The least stable were Gapdh, eukaryotic translation initiation factor 4A2 and Calnexin (Canx). In conclusion, care must be taken when choosing reference transcripts for analysis in qPCR. For in-vitro pharmacological studies, it should not be assumed that 'housekeeping' genes are stable.

Anti-oxidant, in vitro, in vivo anti-inflammatory activity and antiproliferative activity of mefenamic acid and its metal complexes with manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II)

Some new complexes of mefenamic acid with potentially interesting biological activity are described. The complexes of mefenamic acid [Mn(mef)2(H2O)2], 1, [Co(mef)2(H2O)2], 2, [Ni(mef)2(H2O)2], 3, [Cu(mef)2(H2O)]2, 4 and [Zn(mef)2], 5, were prepared by the reaction of mefenamic acid, a potent anti-inflammatory drug with metal salts. Optical and infrared spectral data of these new complexes are reported. Monomeric six-coordinated species were isolated in the solid state for Mn(II), Ni(II) and Co(II), dimeric five-coordinated for Cu(II) and monomeric four-coordinated for Zn(II). In DMF or CHCl3 solution the coordination number is retained and the coordinated molecules of water are replaced by solvent molecules. The anti-oxidant properties of the complexes were evaluated using the 1,1-diphenyl-2-picrylhydrazyl, DPPH, free radical scavenging assay. The scavenging activities of the complexes were measured and compared with those of the free drug and vitamin C. We have explored their ability to inhibit soybean lipoxygenase, β-glucuronidase and trypsin- induced proteolysis. The complex [Mn(mef)2(H2O)2] exhibits the highest antioxidant activity and the highest inhibitory effect against the soybean lipogygenase (LOX), properties that are not demonstrated by mefenamic acid. Their inhibitory effects on rat paw edema induced by Carrageenan was studied and compared with those of mefenamic acid. The complex [Zn(mef)2] exhibited a strong inhibitory effect at 0.1 mmol/Kg B.W. (81.5 ± 1.3% inhibition), superior to the inhibition induced by mefenamic acid at the same dose (61.5 ± 2.3% inhibition). Mefenamic acid and its metal complexes have been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse fibroblast L-929 cell line. The copper(II) complex displays against T24, MCF-7 and L-929 cancer cell lines, IC50 values in a μM range similar to that of the antitumor drug cis-platin and they are considered for further stages of screening in vitro and/or in vivo as agents with potential antitumor activity.

Bilayered chitosan-based scaffolds for osteochondral tissue engineering: Influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor

Osteochondral tissue engineering presents a current research challenge due to the necessity of combining both bone and cartilage tissue engineering principles. In the present study, bilayered chitosan-based scaffolds are developed based on the optimization of both polymeric and composite scaffolds. A particle aggregation methodology is proposed in order to achieve an improved integrative bone–cartilage interface needed for this application, since any discontinuity is likely to cause long-term device failure. Cytotoxicity was evaluated by the MTS assay with the L929 fibroblast cell line for different conditions. Surprisingly, in composite scaffolds using unsintered hydroxyapatite, cytotoxicity was observed in vitro. This work reports the investigation that was conducted to overcome and explain this behaviour. It is suggest that the uptake of divalent cations may induce the cytotoxic behaviour. Sintered hydroxyapatite was consequently used and showed no cytotoxicity when compared to the controls. Microcomputed tomography (micro-CT) was carried out to accurately quantify porosity, interconnectivity, ceramic content, particle and pore sizes. The results showed that the developed scaffolds are highly interconnected and present the ideal pore size range to be morphometrically suitable for the proposed applications. Dynamical mechanical analysis (DMA) demonstrated that the scaffolds are mechanically stable in the wet state even under dynamic compression. The obtained elastic modulus was, respectively, 4.21±1.04, 7.98±1.77 and 6.26±1.04MPa at 1Hz frequency for polymeric, composite and bilayered scaffolds. Bioactivity studies using both a simulated body fluid (SBF) and a simulated synovial fluid (SSF) were conducted in order to assure that the polymeric component for chondrogenic part would not mineralize, as confirmed by scanning electron microscopy (SEM), inductively coupled plasma-optical emission spectroscopy (ICP) and energy-dispersive spectroscopy (EDS) for different immersion periods. The assays were carried out also under dynamic conditions using, for this purpose, a specifically designed double-chamber bioreactor, aiming at a future osteochondral application. It was concluded that chitosan-based bilayered scaffolds produced by particle aggregation overcome any risk of delamination of both polymeric and composite parts designed, respectively, for chondrogenic and osteogenic components that are mechanically stable. Moreover, the proposed bilayered scaffolds could serve as alternative, biocompatible and safe biodegradable scaffolds for osteochondral tissue engineering applications.

Anti-bacterial and cytotoxic properties of plasma sprayed silver-containing HA coatings

Silver-containing hydroxyapatite (HA) coatings have been prepared on titanium substrate by vacuum plasma spraying (VPS) method and anti-bacterial properties of the coatings were examined. Three types of bacteria stains, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, were employed in this test. The results showed that the silver-containing HA coatings exhibited significant anti-bacterial effects against the three bacteria with anti-bacterial ratios higher than 95%. The release of silver ions in the physiological environment ensured excellent anti-bacterial properties of the silver-containing HA coatings. International standard ISO 10993-12 was adopted for cytotoxicity evaluation using fibroblast cell line L929, and it was found that the cytotoxicity for the coatings ranked 0 that showed no cytotoxicity for the coatings. Hemolysis test was processed according to ASTM F 756 standard with anti-coagulated rabbit blood, and the hemolysis ratios of the coatings were below 0.4%, indicating of non-hemolysis for the coatings.

Characterization of surface modified polyester fabric

Woven polyethylene terephthalate (PET) fabric has been used in the construction of vascular grafts and sewing ring of prosthetic heart valves. In an effort to improve haemocompatibility and tissue response to PET fabric, a fluoropolymer, polyvinylidine fluoride (PVDF), was coated on PET fabric by dip coating technique. The coating was found to be uniform and no significant changes occurred on physical properties such as water permeability and burst strength. Cell culture cytotoxicity studies showed that coated PET was non-cytotoxic to L929 fibroblast cell lines. In vitro studies revealed that coating improved haemocompatibility of PET fabric material. Coating reduced platelet consumption of PET fabric by 50%. Upon surface modification leukocyte consumption of PET was reduced by 24%. About 60% reduction in partial thromboplastin time (PTT) observed when PET was coated with PVDF. Results of endothelial cell proliferation studies showed that surface coating did not have any substantial impact on cell proliferation. Overall results indicate that coating has potential to improve haemocompatibility of PET fabric without affecting its mechanical performance.

Plasma Surface Modification of Chitosan Membranes: Characterization and Preliminary Cell Response Studies

Surface modification of biomaterials is a way to tailor cell responses whilst retaining the bulk properties. In this work, chitosan membranes were prepared by solvent casting and treated with nitrogen or argon plasma at 20 W for 10-40 min. AFM indicated an increase in the surface roughness as a result of the ongoing etching process. XPS and contact angle measurements showed different surface elemental compositions and higher surface free energy. The MTS test and direct contact assays with an L929 fibroblast cell line indicated that the plasma treatment improved the cell adhesion and proliferation. Overall, the results demonstrated that such plasma treatments could significantly improve the biocompatibility of chitosan membranes and thus improve their potential in wound dressings and tissue engineering applications.

In vitro evaluation of casein phosphopeptide‐amorphous calcium phosphate as a potential tooth transport medium: viability and apoptosis in L929 fibroblasts

Casein phosphopeptides (CPP) are derived from casein, which accounts for 80% of the total protein in bovine milk . The purpose of this in vitro study was to evaluate the potential use of a CPP-amorphous calcium phosphate (CPP-ACP) preparation as a transport medium for avulsed teeth. L929 fibroblastic cell line was plated in 24-well culture plates. Following incubation, the cells were treated with 10(-3), 10(-4), 10(-6), 10(-8), 10(-12) dilutions of a water-based CPP-ACP paste (Tooth Mousse, GC Corp., Tokyo, Japan). Untreated cells served as controls. The L929 cells were counted at the 1st, 3rd and 7th days. Propidium iodide/acridine orange staining was used to assess apoptosis of treated cells and of the positive control. For each concentration (dilution), statistical analysis of cell survival within time was performed using two-way analysis of variance (ANOVA, P = 0.05). One way ANOVA and Tukey tests were applied to compare the effect of different concentrations on cell survival at each evaluation day (P = 0.05). Except for the 10(-3) and 10(-4) dilutions, all groups demonstrated an increase in cell numbers at days 1 and 3, followed by a decrease at day 7. Irrespective of the increase or decrease in cell viability, time-dependent changes for each dilution group were significantly different. Cells in the 10(-3) and 10(-4) dilution groups demonstrated a rapid apoptotic response. A relatively few number of apoptotic cells were observed in the 10(-6) and 10(-8) dilution groups, while no sign of apoptosis was evident in the 10(-12) dilution group and control. These results suggest that when highly diluted, the tested CPP-ACP preparation may help preserve L929 cell viability in the short term without inducing apoptosis.

Study on the biocompatibility of novel terpolyester poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate‐co‐3‐hydroxyhexanoate)

Terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) containing 5.3 mol % 3-hydroxyvalerate (3-HV) and 10.2 mol % 3-hydroxyhexanoate was obtained via microbial synthesis using recombinant Aeromonas hydrophila. For the first time in vitro biocompatibility of the terpolyester was evaluated in comparison with poly(L-lactic acid), poly (3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Scanning electron microscopy showed whorls-like morphology only on PHBVHHx film surface. Methylthiazol tetrazolium assay demonstrated that PHBVHHx was better than the above four materials in promoting cell attachment and proliferation of fibroblast cell line L929 and osteoblast cell line MC3T3, respectively. Histological study using rabbits proved that PHBVHHx was a fairly harmless implantable biomaterial. Thus, PHBVHHx with an adjustable mechanical properties, combined with its biocompatibility, are in the process of developing into a new generation of bioimplant material.

Lung Delivery Studies Using siRNA Conjugated to TAT(48−60) and Penetratin Reveal Peptide Induced Reduction in Gene Expression and Induction of Innate Immunity

The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.

Photosensitizing Potential of Ciprofloxacin at Ambient Level of UV Radiation

Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV-R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L-929 and NIH-3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2*-) and hydroxyl radical (*OH) was studied under the exposure of ambient intensities of UV-A (1.14, 1.6 and 2.2 mW cm(-2)), UV-B (0.6, 0.9 and 1.2 mW cm(-2)) and sunlight (60 min). The drug was generating 1O2, O2*- and *OH in a concentration and dose-dependent manner. Sodium azide (NaN3) and 1,4-diazabicyclo 2-2-2-octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90-95% O2*- generation. The drug (5-40 microg mL(-1)) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL(-1)) confirms the involvement of ROS in drug-induced lipid peroxidation. The generation of *OH radical was further confirmed by using specific quenchers of *OH such as mannitol (0.5 M) and sodium benzoate (0.5 M). 2'-deoxyguanosine (2'-dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin-sensitized photo-degradation of guanine base. L-929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 microg mL(-1)) under UV-A, UV-B and sunlight, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in NIH-3T3 cell line at higher drug concentration (300 microg mL(-1)) showed a decrease in cell viability by 54%, 56% and 59% under UV-A, UV-B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L-929 cell line were in accordance with MTT assay. The NIH-3T3 cell line showed a higher photosensitizing potential than L-929. The phototoxicity end point shows a time- and concentration-dependent statistically significant (P<0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV-induced photo-peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin-induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.

In vitro effect of oligo-hydroxyalkanoates on the growth of mouse fibroblast cell line L929

The cellular responses to polyhydroxyalkanoates (PHA) degradation products oligo-hydroxyalkanoates (OHAs) are very important factors that control the biocompatibility of these polymers when they are used in tissue-engineering applications. In this study, oligo(3-hydroxybutyrate) (OHB, Mn 2000), oligo(3-hydroxybutyrate- co-4-hydroxybutyrate) (O3HB4HB, Mn 2100, 6 mol% 4HB), oligo(3-hydroxybutyrate- co-3-hydroxyhexanoate) (OHBHHx, Mn 2800, 12 mol% 3HHx) and medium-chain-length oligo(3-hydroxyalkanoates) (OmclHAs, Mn 2300, 2 mol% 3-hydroxyhexanoate (3HHx), 25 mol% 3-hydroxyoctanoate, 71 mol% 3-hydroxydecanoate and 3 mol% 3-hydroxydodecanoate) were prepared as insoluble particles in aqueous solution. Liposomes were employed to encapsulate OHAs and facilitate their transfer into the cytosol. The methylthiazol tetrazolium (MTT) assay, a cell apoptosis study and the flow cytometry were used to evaluate cell viability of mouse fibroblast L929. OHAs in concentration lower than 20 mg/l did not significantly affect cell viability, while OHAs over 40 mg/l reduced cell viability with more cell apoptosis, more cell death, delayed cell cycle and reduced cell proliferation. The cytotoxicity of OHAs decreased with increasing OHAs side chain length. It is proposed that medium-chain-length OHAs containing PHA, such as poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx) and medium-chain-length PHA Poly(3-hydroxyalkanoates) (mcl PHA), are preferred for development and application as PHA-based tissue-engineering biomaterials.