Excipient toxicity and tolerability in self-emulsifying drug delivery systems: insights from cell-based assays

Abstract

The toxicological evaluation of excipients plays a crucial role in the development of SEDDS. This study examined key formulation components to identify critical factors influencing cellular tolerance and supports the design of biocompatible SEDDS. Physicochemical properties of various oils, co-solvents, co-surfactants, non-ionic and charged surfactants formulated in SEDDS were determined via dynamic light scattering, while oxidative stability of selected surfactants was assessed through hydroperoxide quantification. Biological compatibility was evaluated by analyzing hemolytic effects on human erythrocytes and cell viability in CaCo-2 and HEK-293 cells. Metabolic activity and proliferation were additionally measured photometrically via MTT test. Cellular compatibility varied markedly among individual excipients, depending on their chemical structure and formulation role. In lipid-based systems, saturated triglycerides yielded up to sixfold higher viability than free fatty acids. Co-solvent toxicity correlated with lipophilicity and functional groups: isopropanol induced early membrane stress in CaCo-2 cells, while glycerol caused delayed hemolysis after 48  h. A clear structure–activity relationship emerged across surfactant types. PEG-based surfactants outperformed fatty alcohols and sugar-based formulations, which reached IC₅₀ values below 0.01% after 24  h and triggered early proliferation loss. This trend aligned with peroxide levels, as surfactants < 10  mM consistently maintained high CaCo-2 viability and stable IC₅₀ values, exemplified by polyoxyl 40 hydrogenated castor oil. Among zwitterionic surfactants, phosphatidylcholine showed highest biocompatibility, causing only a twofold reduction in hemolytic activity after 48  h, whereas cocoamidohydroxypropyl sulfobetaine induced an 12-fold decrease within 3  h. These findings underscore the role of excipient selection in minimizing cellular stress and adverse drug reactions in oral lipid-based drug delivery.

Introduction

Historically, self-emulsifying drug delivery systems (SEDDS) have evolved from lipid-water systems, which have been the subject of intensive research since the mid-20th century. Early studies on microemulsions demonstrated the potential of thermodynamically stable dispersions with high surfactant and co-solvent content for enhancing the bioavailability of poorly water-soluble active ingredients (Hoar and Schulman, 1943, Constantinides, 1995). Closely related lyotropic liquid crystalline phases, characterized by distinct morphologies (e.g., lamellar or hexagonal), further underscored the structural versatility of amphiphilic excipients in aqueous environments (Singh and Singh, 2024). Despite their scientific relevance, these systems were often limited by the complexity of formulation, reproducibility and long-term stability (Constantinides, 1995, Egito, 2021).

Against this background, SEDDS proved to be a promising further development of earlier microemulsion concepts. Despite their thermodynamic instability, SEDDS are kinetically stable and generate fine oil in water emulsions in gastrointestinal fluids, thereby overcoming solubility barriers (Uttreja, 2025, Rehman, 2022). This conceptual transformation positioned SEDDS as a technologically feasible and clinically attractive formulation option that bridges the gap between fundamental colloid science and modern pharmaceutical applications (Uttreja, 2025, Pouton, 1985). Typically, these so-called “emulsion concentrates” consist of isotropic mixtures of natural or synthetic oils, surfactants as well as optional co-surfactants and/or co-solvents. In current research, focus has been directed toward the role of excipients in SEDDS – not only as functional carriers, but also as potential determinants of safety and tolerability (Furrer, 2013, Sakshi Wakchaure et al., 2024). As the study by Pockle et al. (Pockle, 2023) showed, excipients are not pharmacologically inert at all. They significantly influence the pharmacokinetics of an active ingredient, for example by modulating metabolic enzymes (e.g., CYP450), transporter interactions, or by altering plasma protein binding (Patel et al., 2020).

Especially in SEDDS, certain excipients such as polyethylene glycol derivatives or ionic surfactants can disrupt cell membrane integrity and lead to hemolytic effects (Le-Vinh, 2024). The surface charge of emulsion droplets – which is largely determined by the choice of excipients – influences their binding to plasma proteins, systemic distribution, and cellular uptake. Studies by Hung and Taylor (Hung and Taylor, 1980) as well as Gheorghe et al. (St. Gheorghe et al., 2012) displayed that positively charged droplets, especially quaternary ammonium compounds, exhibit stronger membrane interaction and potentially higher toxicity (Zhang, 2008). In contrast, negatively charged surfactants such as linear alkylbenzene sulfonates are less cytotoxic but can have skin-irritating and ecotoxic effects (Badmus, 2021). Zwitterionic surfactants, on the other hand, are considered biocompatible due to their balanced charge distribution and reduced interaction with cell membranes, resulting in less cellular disruption (Lu et al., 2024, Stengel and PEG vs. zwitterions, 2023).

In addition to their impact on bioavailability and membrane integrity, excipients also affect gastrointestinal transit, active transport, and presystemic metabolism (Martinez, 2022). These systemic effects highlight the need for a comprehensive evaluation of excipient tolerability beyond their technological role. Significantly, there is extensive prior experience with lipid-based excipients in both animal and human studies, which has provided valuable insights into their pharmacological and toxicological behavior (Thomazini, 2024, Nakmode, 2022, Mueller, 1994). In current research, the main focus was on the examination of individual excipients used in SEDDS extensively via early cell-based assays to systematically evaluate excipient-related effects under controlled in vitro conditions.

Briefly, this study investigated how different excipients influence the physicochemical properties of SEDDS and their association with excipient-dependent cellular responses. In order to achieve this, excipients commonly used in such formulations were grouped into six representative test systems (A–F). This composition reflects the major functional categories relevant for SEDDS design, namely oils (A), co‑solvents (B), co‑surfactants (C and D) and nonionic surfactants (E) as well as ionic surfactants (F). In line with the central role of the oil phase in SEDDS, the evaluation was initiated with an oil screening as the primary component and subsequently extended stepwise to further excipient categories. This sequential approach ensured a systematic progression from the core formulation element towards increasingly complex excipient classes.

To comprehensively assess cellular compatibility, three complementary test methods were applied: a hemolysis assay to evaluate membrane damage in human erythrocytes, a cytotoxicity test on CaCo-2 and HEK-293 cell lines to assess cell viability and a MTT proliferation assay to detect potential effects on cell division. These assays were selected to capture both direct membrane effects and broader cellular responses, thereby providing a rationale for assessing excipient-related cytotoxicity in vitro. Formulations were incubated for defined periods of 3, 24 and 48 h to monitor both immediate and delayed cellular responses.

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Materials

Tricaprylin (Captex® 8000), caprylic/capric triglyceride/C8 dominant (Captex® 355), caprylic/capric triglyceride/ balanced C8/C10 ratio (Captex® 300), glyceryl caprylate/caprate (Capmul® MCM) and propylene glycol caprylate (C8), mixed mono-/diester (Capmul® PG-8) were kindly provided by Abitec (Janesville, USA). Triton X-100 solution, glycerin, benzyl alcohol, propylene glycol, oleyl alcohol, polyoxyl 35 castor oil (Kolliphor® EL), didodecyldimethylammonium bromide, and sodium dodecylbenzenesulfonate were obtained from Sigma-Aldrich (Vienna, Austria). Phosphatidylcholine (Lipoid® S 100) was provided by Lipoid GmbH (Ludwigshafen, Germany). Oleic acid and isopropyl alcohol were obtained from VWR (Vienna, Austria). Cetyltrimethylammonium bromide was supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Ethanol was obtained from DonauChem (Vienna, Austria). Propylene glycol monocaprylate (C8; Capryol® 90), caprylic/capric triglyceride (Labrafac® Lipophile WL 1349), glyceryl oleate (Peceol®), diethylene glycol monoethyl ether (Transcutol®) and oleoyl polyoxyl-6 glycerides (Labrafil® M 1944) were donated by Gattefossé (Saint-Priest, France). Polyglyceryl-4 Caprate (Tegosoft® PC 41), Polyglyceryl-6 caprylate (and) polyglyceryl-4 caprate (TegoSolve® 90 MB) and polyglyceryl-6 caprylate, −3 cocoate, −4 caprate, −6 ricinoleate (TegoSolve® 61 MB) were donated by Evonik Nutrition & Care GmbH (Essen, Germany). Polyethylene glycol (PEG 400) was obtained from Gatt-Koller (Absam, Austria). Polyoxyl 40 hydrogenated castor oil (Kolliphor® RH 40), polyoxyl 15 hydroxystearate (Solutol® HS 15) and alkyl polyglucoside (lauryl glucoside) (Plantapon® LGC Sorb) were ordered from BASF (Ludwigshafen, Germany). Sodium decanoate was kindly provided by TCI EUROPE N.V. (Zwijndrecht, Belgium). Alkylpolyglucosides (C8–C10; Multitrope® 1620) was supplied by Croda (Nettetal, Germany). Sodium deoxycholate was obtained from AppliChem GmbH (Darmstadt, Germany). Ethyllauroyl arginate and cocoamidohydroxypropyl sulfobetaine were obtained from Biosynth Laboratories Ltd. (Billingham, UK). Sucrose laurate (sucrose ester) (Sisterna® L70-C) was purchased from Sisterna B.V. (Roosendaal, Netherlands). MEM Eagle® was sourced from PAN-Biotech (Aidenbach, Germany), while resazurin sodium salt and Pierce Quantitative Peroxide Assay Kit were bought from Thermo Fisher Scientific (Waltham, MA, USA). The EZ4U non-radioactive cell proliferation and cytotoxicity kit was obtained from Biomedica (Vienna, Austria)..

Marlene Ramona Schmidt, Magdalena Ender, Melanie Lena Ebert, Khush Bakhat Afzal, Astrid Dagmar Bernkop-Schnürch, Andreas Bernkop-Schnürch, Excipient toxicity and tolerability in self-emulsifying drug delivery systems: insights from cell-based assays, International Journal of Pharmaceutics, 2026, 126646, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2026.126646.