Enhancement of chlorogenic acid uptake and transport across co-cultured Caco-2/HT29-MTX-E12 cell membranes

Abstract

Background: Chlorogenic acid (CQA) is one of the ester derivatives of quinic acid, which is considered as one of the major bioactive compounds obtained from fruit and coffee with excellent antioxidant and anti-inflammatory effects. CQA is classified as a Class III drug due to its high hydrophilicity and low permeability (oral bioavailability, 33%). Therefore, the aim of this study is to conduct the preformulation study of CQA and to develop and characterise CQA loaded-solid lipid nanoparticles (CQA-SLNs) formulation to enhance the permeability of CQA across biological membranes. The intestinal absorption mechanism of CQA was also investigated with/without SLNs in the absence or presence of variable agents. Method: Physicochemical properties of CQA were investigated before formulation design, including morphology, Fourier-transform infrared spectroscopy, thermal analysis, forced degradation, and cytotoxicity. A reliable and sensitive HPLC method was developed and validated for the determination of CQA. The CQA-SLNs were prepared by the double emulsion method. A 2-level 3-factor factorial design was employed to optimise the SLN formulation using Design-Expert® software. Optimised CQA-SLNs were characterised for particle size, zeta potential, entrapment efficiency (EE), in vitro drug release, and stability. The uptake and transport mechanisms were investigated in the absence or presence of variable inhibitors or enhancers using Caco-2 or co-cultured Caco-2/HT29-MTX-E12 cell culture models. Result: A reliable HPLC method for CQA was developed with a retention time at 4.03 min. The calibration curve was linear over a concentration range of 1-100 μg/mL (r2>0.9996). Intraday and inter-day variabilities were less than 0.69% and 2.67%, respectively. The accuracy was in the range of 99.12-100.20%. Forced degradation studies showed CQA was stable in acidic, oxidative, high temperature and light conditions after 7 days. Significant degradation was found in basic conditions, where only 3.73 ± 0.17% remained after 1 minute. The optimised CQA-SLNs formulation demonstrated uniform morphology, with particle size (528 ± 16 nm), polydispersity index (0.39 ± 0.03), zeta potential (+27 ± 0.51 mV), EE (30.18 ± 0.33%), and acceptable stability at 4°C. In vitro release studies showed sustained release over 24 h following the Higuchi model. CQA, CQA-SLNs exhibited negligible cytotoxicity towards Caco-2 cells. Additionally, confocal microscopy study confirmed the uptake of CQA-SLNs into cells. CQA cellular uptake was significantly decreased by chlorpromazine (clathrin-mediated endocytosis inhibitor) and increased by protamine sulphate (adsorptive-mediated endocytosis inhibitor), while CQA-SLNs uptake was significantly reduced by protamine sulphate, chlorpromazine, and at 4°C incubation. The transport of CQA was significantly enhanced by EDTA, sodium decanoate, verapamil and when encapsulated in SLNs. CQA-SLNs demonstrated two-folds increase in the apparent permeability coefficient (Papp) from 0.52 ± 0.103 × 10-6 to 1.14 ± 0.023 × 10-6 cm/s. Conclusion: Preformulation parameters of CQA were thoroughly studied. A CQA-loaded SLN system was developed, optimised and characterised. The mechanisms of CQA uptake and transport were investigated in the absence or presence of formulation and variable agents using Caco-2 and HT29-MTX-E12 co- cultured cell models.