Cytotoxicity of prostaglandin analog eye drops preserved with benzalkonium chloride in multiple corneoconjunctival cell lines

Masahiko Ayaki1, Atsuo Iwasawa21Department of Ophthalmology, Saitama National Hospital, Wako, 2Department of Clinical Pathology, Showa University Fujigaoka Hospital, Yokohama, JapanPurpose: This study evaluated the cytotoxicity of five prostaglandin analog ophthalmic ­solutions on four ocular surface cell lines, ie, Chang (human conjunctiva), SIRC (rabbit cornea), RC-1 (rabbit cornea), and BCE C/D-1b (bovine cornea).Methods: Cell viability was measured by neutral red and MTT assays in cells treated for 10, 30, or 60 minutes with various doses of prostaglandins (undiluted, and 2- and 10-fold dilutions). The number of cell lines with viability ≥50% in the presence of selected dilution of the drug (CVS50) was used for comparison. In addition, 24 cell viability comparisons (four cell lines, two assays, and three exposure times) were made between latanoprost (Xalatan®) and each other solution at each dose. A comparison between the newly introduced tafluprost (Tapros®) with 0.01% benzalkonium chloride was also made.Results: The order of cell viability determined by CVS50 was Travatan Z® (travoprost with the SofZia system)> Tapros ≥ Travatan® (travoprost) = Xalatan > Rescula® (unoproston). This was consistent with the results of direct comparisons between Xalatan and the other drugs. There was no clear difference in cell viability between Tapros and benzalkonium chloride.Conclusions: Use of two assays, multiple cell lines, and various dilutions and exposure times provided a unique evaluation of cytotoxicity among ophthalmic solutions. CVS50 was useful for comparison of the cell viability of the solutions.Keywords: glaucoma, cornea, cell viability scor

Toxicity of antiglaucoma drugs with and without benzalkonium chloride to cultured human corneal endothelial cells

Masahiko Ayaki1, Atsuo Iwasawa2, Yoichi Inoue31Department of Ophthalmology, Saitama National Hospital, Wako, Japan; 2Life Particle Interaction Engineering Creation, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan; 3Department of Ophthalmology, Olympia Eye Hospital, Tokyo, JapanPurpose: The toxicity of antiglaucoma medications to ocular surface cells has been evaluated extensively; however, the toxicity to corneal endothelial cells (CECs) remains elusive. Our aim is to evaluate the toxicity of antiglaucoma medications to CECs using an in vitro toxicity assay.Methods: Primary cultures of human (H) CECs derived from eye bank specimens were established. Following exposure of HCECs to test solutions for 10, 30, or 60 minutes, or 48 hours, we measured cell viability using a WST-1 assay. Test solutions were diluted in culture media and included 0.5% Timoptol®, preservative-free 0.5% timolol maleate, 1% Trusopt®, preservative-free 1% dorzolamide, Travatan®, Travatan Z®, Xalatan®, and benzalkonium chloride (BAK). To assess cell viability, the value of the test culture well after treatment was expressed as a percentage of that of the control well. Toxicity of each solution was compared using the cell viability score (CVS).Results: After exposure to 10-fold dilutions of test solutions for 48 hours, HCEC viabilities were 48.5% for 0.5% Timoptol, 80.9% for preservative-free 0.5% timolol maleate, 47.0% for 1% Trusopt, 71.7% for preservative-free 1% dorzolamide, 55.5% for Travatan, 88.5% for Travatan Z, and 52.5% for Xalatan. Exposure to test solutions diluted 100-fold or more resulted in HCEC viabilities > 80%, with the exception of preservative-free 1% dorzolamide, which resulted in a viability of 72.0% at a dilution of 100-fold. Based on CVS, the order of cell viability was Travatan Z ≥ preservative-free timolol maleate = preservative-free dorzolamide > 0.5% Timoptol = 1% Trusopt > Travatan ≥ Xalatan. Assessment of the combined effect of drug and BAK revealed that latanoprost reduced the toxicity of BAK.Conclusion: Antiglaucoma eye drops produced HCEC toxicity that appeared to depend on the presence of BAK. Because dilution of the antiglaucoma solutions resulted in markedly lower HCEC toxicity, HCEC damage due to antiglaucoma medication may occur only in rare cases. The CVS was useful for comparison of the toxicity of the drugs.Keywords: cell viability score, eye drop, preservative

Fungicidal Action of Hydroxyl Radicals Generated by Ultrasound in Water

It is well known that hydroxyl radicals are generated by ultrasound in water. This study with an electron spin resonance spin-trapping technique showed that hydroxyl radical generation was positively correlated with ultrasound duration and water temperature. The clear fungicidal action against Trichophyton spp. evident by studying cultured cells and the degradation of cytoplasmic and surface structures observed by transmission and scanning electron microscopy suggest that ultrasound in hot water is effective for sterilization of dermatophyte contamination and could be effective for the treatment of tinea infection

Ocular Surface Cytotoxicity and Safety Evaluation of Tafluprost, a Recently Developed Anti-Glaucoma Prostaglandin Analog

In vitro cytotoxicity of tafluprost, which is the most recently developed anti-glaucoma prostaglandin (PG) analog, in ocular surface cells is addressed in comparison with other PG analogs. Irrespective of cell lines and models, the cytotoxicity of anti-glaucoma PG eyedrops was primarily related to the concentration of benzalkonium chloride (BAK) contained in the eyedrops as a preservative. Accordingly, preservative-free tafluprost was apparently less cytotoxic than BAK-preserved PG analogs. Furthermore, our study for cytotoxicity assays on ocular cells, conducted by comprehensive investigations covering a variety of concentrations and treatment times, which is termed the cell viability score (CVS) system, demonstrated that 0.001% BAK-preserved tafluprost was not cytotoxic, and suggested that tafluprost may even reduce the cytotoxic effect of BAK. It has been reported that adverse reactions associated with tafluprost in healthy human volunteers and patients with glaucoma include conjunctival hyperemia, eyelid pigmentation, eyelash bristles, and deepening of upper eyelid sulcus. Nonetheless, most clinical studies have demonstrated that not only preservative-free tafluprost but also BAK-preserved tafluprost is well tolerated and safe in patients with glaucoma and ocular hypertension

Plasma Gas Temperature Control Performance of Metal 3D-Printed Multi-Gas Temperature-Controllable Plasma Jet

The aim of the study was to design and build a multi-gas temperature-controllable plasma jet that can control the gas temperature of plasmas with various gas species, and evaluated its temperature control performance. In this device, a fluid at an arbitrary controlled temperature is circulated through the plasma jet body. The gas exchanges heat with the plasma jet body to control the plasma temperature. Based on this concept, a complex-shaped plasma jet with two channels in the plasma jet body, a temperature control fluid (TCF) channel, and a gas channel was designed. The temperature control performance of nitrogen gas was evaluated using computational fluid dynamics analysis, which found that the gas temperature changed proportionally to the TCF temperature. The designed plasma jet body was fabricated using metal 3D-printer technology. Using the fabricated plasma jet body, stable plasmas of argon, oxygen, carbon dioxide, and nitrogen were generated. By varying the plasma jet body temperature from −30 °C to 90 °C, the gas temperature was successfully controlled linearly in the range of 29–85 °C for all plasma gas species. This is expected to further expand the range of applications of atmospheric low temperature plasma and to improve the plasma treatment effect

Influence of Controlling Plasma Gas Species and Temperature on Reactive Species and Bactericidal Effect of the Plasma

In this study, plasma gas species and temperature were varied to evaluate the reactive species produced and the bactericidal effect of plasma. Nitrogen, carbon dioxide, oxygen, and argon were used as the gas species, and the gas temperature of each plasma was varied from 30 to 90 °C. Singlet oxygen, OH radicals, hydrogen peroxide, and ozone generated by the plasma were trapped in a liquid, and then measured. Nitrogen plasma produced up to 172 µM of the OH radical, which was higher than that of the other plasmas. In carbon dioxide plasma, the concentration of singlet oxygen increased from 77 to 812 µM, as the plasma gas temperature increased from 30 to 90 °C. The bactericidal effect of carbon dioxide and nitrogen plasma was evaluated using bactericidal ability, which indicated the log reduction per minute. In carbon dioxide plasma, the bactericidal ability increased from 5.6 to 38.8, as the temperature of the plasma gas increased from 30 to 90 °C. Conversely, nitrogen plasma did not exhibit a high bactericidal effect. These results demonstrate that the plasma gas type and temperature have a significant influence on the reactive species produced and the bactericidal effect of plasma

Photolysis of Hydrogen Peroxide, an Effective Disinfection System via Hydroxyl Radical Formation▿

The relationship between the amount of hydroxyl radicals generated by photolysis of H2O2 and bactericidal activity was examined. H2O2 (1 M) was irradiated with laser light at a wavelength of 405 nm to generate hydroxyl radicals. Electron spin resonance spin trapping analysis showed that the amount of hydroxyl radicals produced increased with the irradiation time. Four species of pathogenic oral bacteria, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Enterococcus faecalis, were used in the bactericidal assay. S. mutans in a model biofilm was also examined. Laser irradiation of suspensions in 1 M H2O2 resulted in a >99.99% reduction of the viable counts of each of the test species within 3 min of treatment. Treatment of S. mutans in a biofilm resulted in a >99.999% reduction of viable counts within 3 min. Other results demonstrated that the bactericidal activity was dependent on the amount of hydroxyl radicals generated. Treatment of bacteria with 200 to 300 μM hydroxyl radicals would result in reductions of viable counts of >99.99%