Cytotoxic Properties of Tyloxapol
Jung-hua Steven Kuo,
and Hsuan Wen Chiu
Received September 2, 2005; accepted February 28, 2006
Purpose. Tyloxapol, a viscous polymer of the alkyl aryl polyether alcohol type, is classified as a nonionic
surfactant and is widely used in biomedical applications. Although tyloxapol has been reported to be
cytotoxic in various cell lines, there is no published information about its possible mechanisms of cell
death. Hence, the objective of this study was to determine whether tyloxapol causes apoptosis or
necrosis. These data could be helpful for a better understanding of the action of tyloxapol in cellular
Methods. RAW 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated
with tyloxapol, and the activity of dehydrogenases in those cells, an indicator of cell viability, was
assessed. The cell morphology changes induced by tyloxapol treatment were detected using propidium
iodide nuclear staining. The hallmarks of apoptotic cells were characterized using DNA fragmentation
assays, DNA fluorescence staining, and then flow analysis.
Results. Tyloxapol treatment produced dose- and time-dependent cytotoxicity. Tyloxapol treatment
damaged RAW 264.7 cells more than it damaged NIH/3T3 cells. All the cells exposed to tyloxapol
showed some morphological features of apoptosis, such as chromatin condensation and cell shrinkage.
Typical apoptotic ladders were observed in DNA extracted from tyloxapol-treated cells. Flow
cytometric analysis revealed an increase in the hypodiploid DNA population (sub-G1), indicating that
DNA cleavage occurred after tyloxapol treatment. In addition, we showed that pretreating cells with
zVAD-fmk, a general caspase inhibitor, did not prevent tyloxapol-induced apoptosis. The cytotoxicity of
tyloxapol can be reduced by adding a nontoxic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine to
attenuate the interaction of tyloxapol with the cell membrane.
Conclusions. Our results indicate that tyloxapol induces apoptosis in RAW 264.7 and NIH/3T3 cells.
These data provide a novel insight into the cytotoxic action of tyloxapol at the molecular level.
KEY WORDS: apoptosis; cytotoxicity; surfactant; tyloxapol.
Tyloxapol (Triton WR-1339) is a nonionic surfactant
oligomer widely used in biomedical applications in vitro and
in vivo, e.g., in contact-lens detergent, mucolytic agents for
pulmonary diseases, inflammatory modulators for endotoxin-
induced activations, components for drug-delivery systems
and synthetic-lung surfactants, a model of hyperlipidemic
atherogenesis, and a powerful up-regulator of dendrimer-
mediated transfection (1Y8). Recently, in vitro biomedical
applications for tyloxapol have been used to prevent
reactions to endotoxin-induced activations in immune cells
(mostly macrophages) (3,9,10). Also, tyloxapol has been used
to augment nonviral gene transfer in a variety of eukaryotic
cells, especially primary cells [e.g., human lung (NHBE) and
porcine vascular endothelial (YPE) cells] that are difficult to
transfect (5). Surfactants are known to interact with cell
membranes, and tyloxapol has been reported to be toxic in
epithelial and red blood cells (4,11). Also, the risk of
pulmonary hemorrhage has been reported to increase after
intratracheal treatment with colfosceril palmitate HSE
(dipalmitoylphosphatidylcholine; Exosurf Neonatal; Glaxo-
SmithKline, Middlesex, UK) in extremely low-birth-weight
infants with respiratory distress syndrome. This risk may be
related to the cytotoxicity of tyloxapol (11). However, the
mechanism it uses to induce cell death remains unknown.
The two major causes of cell death are necrosis and
apoptosis (12). Necrosis is related to an inflammatory and a
degenerative process (13). Cells undergoing necrosis charac-
teristically illustrate mitochondrial swelling, lose membrane
integrity, turn off metabolism, and release a cytoplasmic
component that stimulates an inflammatory response. Apo-
2006 Springer Science + Business Media, Inc.
Pharmaceutical Research, Vol. 23, No. 7, July 2006 (
Graduate Institute of Pharmaceutical Science, Chia Nan University
of Pharmacy and Science, 60 Erh-Jen Road, Sec. 1, Jen-Te, Tainan
Department of Microbiology and Immunology, Medical College of
Chung Shan Medical University, 110, Sec. 1, Jianguo Road,
Department of Biotechnology, Chia Nan University of Pharmacy
and Science, 60 Erh-Jen Road, Sec. 1, Jen-Te, Tainan 717, Taiwan.
To whom correspondence should be addressed.(e-mail:kuojunghua@