Gastrointestinal mucoadhesion, absorption and biodistribution of orally administered chitosan-coated amphotericin B nanostructured lipid carriers (NLC) formulation

AmpB is a polyene macrolide with broad spectrum antifungal activity. Oral delivery of AmpB is desirable because it provides a more patient-friendly mode of administration compared to the current delivery approach akin with the present AmpB formulation. However, none of the attempts to realise this desirability has materialised as yet due to the challenging physicochemical characteristics of AmpB. The advantages of encapsulating a drug within NLCs are well-documented and coupled with the above goal of delivering AmpB orally, a logical addition would be to employ a gastrointestinal retention modality to the NLCs in order to delay transit and maximise absorption. This is in line with the present work which focuses on the formulation and characterisation of a mucoadhesive AmpB-loaded NLC system. The particle size of the uncoated AmpB NLC was 163.1 ± 0.7 nm with a narrow PDI value of 0.19 ± 0.01 and ζ of -42.4 ± 1.2 mV. Upon adsorption of the chitosan coating, the resulting ChiAmpB NLC observed a two-fold increase in particle size, to 348.0 ± 12 nm and reversion of the ζ towards positivity, +24.3 ± 1.4 mV. The adsorption of the chitosan coating was further ascertained through FTIR-ATR analysis which presented two distinctive peaks akin to amide I and II from the chitosan at 1635 and 1539 cm-1 respectively. The NLCs were spherical and well-dispersed under the STEM. The aggregation states and EE of AmpB were dependent on the mode of incorporation of the AmpB during the formulation process. Incorporation of undissolved AmpB during the initial stage of formulation resulted in a higher EE, 83.4 ± 0.7 % and exhibited safer configuration compared to NLC prepared with dissolved AmpB incorporated the final phase of formulation. Furthermore, at 50 mg loading, the ChiAmpB NLC exhibited slow and sustained release of AmpB and with minimal changes in its physical properties upon exposure to different pH conditions compared to the uncoated formulation. The particle size of ChiAmpB NLC remained unchanged upon incubation at pH 1.2 for 2 hours (p = 0.138), whereas AmpB NLC demonstrated 3.8-fold increase in particle size. In contrast to AmpB NLC, ChiAmpB NLC exhibited a higher retention of of AmpB upon exposure to pH 5.8 to 6.8, showing 63.9 ± 2.8 and 56.1 ± 1.8 %, respectively. The mucoadhesion properties of ChiAmpB NLC were evaluated in porcine mucin and based on i) turbidimetric assay and ii) changes in ζ values. ChiAmpB NLC showed positive mucoadhesion results in both assays by virtue of electrostatic interactions with mucin. The ex vivo mucoadhesive study further confirmed the mucoadhesion properties of ChiAmpB NLC with 84.2 ± 5.1 % bound nanoparticles to the rat intestinal tissue, 28.4 % higher than the uncoated AmpB NLC. The antifungal property of AmpB against Candida albicans was enhanced through ChiAmpB NLC and AmpB NLC, showing lower fungal viabilities at low concentration of AmpB. There were no signs of any acute haemolytic effects in both developed formulations in contrast to pure AmpB and Amphotret® formulations. Moreover, the cytotoxicity assay also revealed that the ChiAmpB NLC was relatively non-toxic to HT-29 cells as 99.1 ± 13.4 % of the cells remained viable upon 24-hour incubation. Orally administered ChiAmpB NLC demonstrated a two-fold increase in the AUC0-∞ values as compared to the uncoated AmpB NLC and Amphotret® via the same route of administration. The enhancement in systemic bioavailability of the AmpB following oral administration suggests the possibility of prolonged retention at the small intestine through the mucoadhesive effect and a subsequent absorption through the lymphatic pathway. The ChiAmpB NLC formulation presents potentially a lower risk of nephrotoxicity with higher accumulation in the liver and spleen thus, the limitations of the current formulation of AmpB have not only been addressed but also opens up the possibility of utilising this approach as a targeting strategy to the desired organs for the treatment of visceral leishmaniasis disease.