Purpose: Lipid-based self-emulsifying drug delivery system (SEDDS) has been widely explored to administer poorly water soluble drugs in a solubilized form along with its potential for enhanced intestinal absorption via surfactant induced membrane fluidity and associated permeability changes. Medium chain lipid-surfactant systems are widely used in this context due to their capability of forming stable self-emulsifying microemulsions.However,literature reports suggest that the medium chain lipids above a certain concentration can cause local toxicity of the intestinal epithelia. Long chain lipids are a better alternative to avoid epithelial cytotoxicity of lipid based formulations given the fact that majority of the endogenous and dietary lipids are long chain glycerides. However,there are no reports available in the literature showing systematic cytotoxicity study of self-emulsifying formulations composed of long chain lipids.
Differentiated caco-2 cells are very similar to normal enterocytes morphologically and are extensively used as in vitro simulation model to predict in vivo intestinal drug absorption.The aim of the present study was to carry out a systematic evaluation of the cytotoxicity of commonly used long chain lipid excipients and formulations in a wide range of concentrations on caco-2 cells of varying maturity.
Methods: Capmul GMO (glyceryl monooleate; GMO), Captex GTO (glyceryl trioleate; GTO), and Kolliphor EL (KEL) were used as the long chain monoglyceride, long chain triglycerides and surfactant, respectively, in the development of lipid-based self-emulsifying formulations or preconcentrates. Each test lipid and surfactant was mixed in the following ratios (v/v): (1) 30% KEL and 70% GMO; (2) 30% KEL, 52.5% GMO and 17.5% GTO; (3) 30% KEL, 35% GMO and 35% GTO. Neat KEL, a mixture of KEL:HBSS (30:70) and each of the above preconcentrate was diluted using HBSS to obtain 0.25%, 0.5%, 1%,2.5% and 5% v/v concentrations. Each diluted concentration was characterized for particle size at 0, 60 and 120 min time points in triplicate using the DelsaNano C particle size analyzer (dynamic light scattering technique) at 25°C.
The cytotoxicity assessment of each preconcentrate was carried out using the MTT Assay.20000 caco-2 cells per well were cultured for the designated durations (1-day, 5-days and 21 days). The cells were treated with test lipid formulations at concentrations of 0.25%, 0.5%, 1%, 2.5% and 5% (v/v) in culture medium for two hours. Cells incubated with only culture medium (no lipid) served as control and were considered 100% viable. After two hours of incubation the treated cells were subjected to MTT Assay.
Results: The particle size analysis indicated that there was no significant difference in the particle size of the formulations at 0 min, 60 min and 120 min. The particle size of aqueous dispersions increased as the concentration of preconcentrate increased from 0.25% to 5% v/v. The emulsions formed showed a particle size in the range of 200 nm to 1700 nm depending on the formulation composition and concentration. Cytotoxicity of lipid-based formulations was influenced by the maturity of caco-2 cells and formulation composition. One-day culture was most sensitive to formulation exposure. When cultured for 5 days, viability of caco-2 cells was remarkably improved. The formulation with 30:35:35 (%v/v) KEL: GMO: GTO was found to be non-toxic at as high as 5%v/v concentration. It was expected that the 21-day monolayer culture would be even more resistant to cytotoxic effects. This was observed to a certain extent, however, the formulation containing 30:35:35 (%v/v) KEL:GMO:GTO which was found to be non-toxic on 5-day cultured caco-2 cells even at 5%v/v showed decreased viability on 21-day caco-2 cell monolayer at 2.5%v/v and 5%v/v concentrations. This indicates a possible interaction of the formulation with tight junctions of the differentiated cells, leading to greater cellular damage. Further studies are being carried out to investigate this interaction and propose a mechanism of toxicity of these formulations.
According to the cytotoxicity data of medium chain lipid formulations on 21-day caco-2 cell culture reported from a previously published study from our lab, medium chain lipid formulations (containing KEL, Capmul MCM (glyceryl monocaprylate) and Captex 355 EP/NF (glyceryl tricaprylate) at 30:52.5:17.5 and 30:35:35 (%v/v)) showed less than 60% cell viability at 0.5% v/v concentrations, whereas long chain lipid formulations showed close to 80% cell viability even at 1% v/v concentration and up to 60% viability at as high as 5% v/v concentrations, highlighting the fact that long chain lipids themselves are not only well tolerated but also better mitigate the surfactant induced cellular toxicity.
Conclusion: Maturity of caco-2 cells rendered significant resistance to cytotoxicity. The monolayers with 21-day maturity are more relevant to in vivo conditions and appeared to be a more accurate in vitro model for cytotoxicity assessment compared to 1-day and 5-day cultures grown on plastic surfaces. Long chain lipid based formulations developed in this study were found to be safe and well tolerated even at high concentrations. Thus, they may serve as a better alternative for the development of SEDDS compared to medium chain lipids
Pengli Bu– St. John's University
Ankita Shah– Post Doctoral Research fellow, St. John's University
Xingguo Cheng– Professor, St. John's University
Abu Serajuddin– Professor, St Johns University, New York