Development and evaluation of meropenem encapsulated nanostructured lipid carriers as an antimicrobial treatment of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative bacterium which most commonly causes opportunistic infections in immunocompromised individuals including those with cystic fibrosis. Meropenem is a carbapenem antibiotic with a beta lactam structure which is recommended as a treatment for meropenem-susceptible P. aeruginosa infections P. aeruginosa has developed multiple resistance mechanisms including resistance to beta lactam antibiotics such as meropenem.
Nanoparticles can be used as a targeted treatment therapy by utilizing a controlled delivery and release of active pharmaceutical ingredient at a specific target site. Therefore, encapsulating antibiotics, such as meropenem, which has high renal elimination, a short half-life and is unstable in aqueous solutions, could provide an alternative drug delivery pathway for treatment of sensitive strains of P. aeruginosa, improving pharmacokinetic outcomes.
Meropenem-encapsulated nanostructured lipid carriers (ME-NLCs) were formulated using Sodium Cholate as a surfactant, Dynasan 114 as a solid lipid, Capryol 90, oleic acid, lipoid E80 and lipoid S75 as liquid lipids, and Kolliphor HS 15 as a solubiliser. A hot homogenisation method followed by probe sonication was used for preparation of NLCs. Optimisation of product parameters (surfactant, liquid and solid lipids, solubiliser and drug concentration) and process parameter (sonication time) was carried out to obtain NLCs with desired physicochemical properties in terms of particle size, polydispersity index, zeta potential and drug entrapment. Increasing the concentration of surfactant sodium cholate resulted in an increase in particle size (<200nm) and poly dispersity index (<0.3) of the NLCs. Similarly, the loading of meropenem in NLCs resulted in an increase of particle size but interestingly did not affect the PDI. Meropenem-loaded NLCs produced a particle size of 178.8nm , poly dispersity index 0.283 and zeta potential -31mV.
A high performance liquid chromatography (HPLC) method was developed and validated to quantify meropenem in the NLCs formulations and drug encapsulation was calculated to be 85.72%. Additionally in vitro drug dissolution studies of the ME-NLCs revealed an initial fast release of 30% of the drug in the first thirty minutes and a sustained release of drug from NLCs with 70% of the drug being released for 8 hours which then remained steady until 24 hours.
Reference isolates of P. aeruginosa ATCC 27853 were tested for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using – ISO-20776-1 method recommended by EUCAST - using the ME-NLCs. The minimum inhibitory concentration for ME-NLC for P. aeruginosa was 2 g/ml. The ME-NLCs was bactericidal at a concentration of 8 g/ml, compared to free drug which has a MIC of 0.5 g/ml and MBC of 4 g/ml.
Interactions between ME-NLCs and P. aeruginosa cells were investigated using fluorescence microscopy. Morphological changes were observed in the bacterial cells which showed an increase in the number of elongated rod-shaped cells.
The use of meropenem encapsulated NLCs may provide an alternative drug delivery system with the potential to optimise therapeutic dosage without increasing nephrotoxicity.