Antioxidant activity of Tween-20 and Tween-80 evaluated through different in-vitro tests Renato Pérez-Rosés a , Ester Risco a,b , Roser Vila a , Pedro Peñalver c and Salvador Cañigueral a a Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, b Phytonexus S.L., Carlet (València) and c Lidervet S.L., Tarragona, Spain Keywords DPPH • ; myeloperoxidase; polysorbates; ROS; Tween Correspondence Salvador Cañigueral, Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona. Av. Joan XXIII, s/n. E-08028 Barcelona, Spain. E-mail: s.canigueral@ub.edu Received February 25, 2014 Accepted November 2, 2014 doi: 10.1111/jphp.12369 Abstract Objectives This study aimed to evaluate the possible antioxidant activity of Tween-20 and Tween-80, two amphipathic nonionic surfactants commonly used as solubilizers and stabilizers, whose pharmacological effects have been ignored to a large degree. Methods Antioxidant activity was investigated in vitro measuring the scavenging activity on the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • ), the pro- duction of reactive oxygen species (ROS) in activated human neutrophils using flow cytometry and the myeloperoxidase (MPO) inhibitory activity. Key findings Tween-20 and Tween-80 did not show scavenging activity on DPPH • , while produced a decrease of the ROS production in human neutrophils, being Tween-20 more active than Tween-80. Moreover, Tween-80 and Tween-20 were found to significantly stimulate MPO enzymatic activity. Conclusions Our findings raise concerns with regard to the indiscriminate use of Tween-20 and Tween-80 in clinical and laboratory testing, since they could influ- ence the results that are assigned to the tested substance. Introduction Nonionic surfactants, such as those of the Tween group (polysorbates), have a wide application in preclinical and clinical testing, since they serve as emulsifiers for lipophilic substances. Nonionic surfactants have been shown to be less toxic than ionic surfactants to biological membranes. [1] Additionally, being more hydrophobic, nonionic surfactants also possess greater capacity to dissolve poorly soluble drugs, are very efficient emulsifiers and can be used in self-emulsifying drug delivery systems. Tween-80 and Tween-20 have similar structural and functional prop- erties. Structurally, both have two nonionic moieties (Figure 1), one hydrophilic (polyethoxylated sorbitol) and another hydrophobic (oleyl or lauryl groups). In aqueous solutions, these molecules tend to self-aggregate and, above the critical micellar concentration, they form colloidal par- ticles, spherical micelles which are lipophilic inside and hydrophilic outside. The size of the particles varies between 5 nm and 25 nm depending on the actual physicochemical properties of the medium. Due to their amphiphilic nature, they can reduce the interfacial tension of solvent systems, and disperse a variety of immiscible materials. Medical application of Tween-20 and Tween-80 is gener- ally recognized as safe. They are extensively used as pharma- ceutical surfactants in different oral and parenteral delivery systems. Its intravenous application is approved by the European Medicines Agency as well as the US Food and Drug Administration. [2] Surprisingly, little is known about how surfactants affect organisms or enzymatic processes. The prevailing notion is that beyond a critical level, surfactants are toxic simply due to their deleterious effect on biomembranes. However, more subtle effects have been reported for Tween surfactants. [3] Several nonionic surfactants have been shown to inhibit transporters, and examples of nonionic surfactants with activity on various efflux pumps include Tweens. [4,5] Tween-80 decreases conca- navalin A-induced proliferative response in human lym- phocytes, and recent studies have revealed that Tween-80 And Pharmacology Journal of Pharmacy Research Paper © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 666–672 666