Nanoencapsulated antimicrobials to fight Pseudomonas aeruginosa respiratory infections in cystic fibrosis patients: a promising strategy

Abstract

P. aeruginosa is one of the major opportunistic pathogen colonizing the respiratory tract of cystic fibrosis (CF) patients and causing chronic airways infection. Once P.aeruginosa established chronically in the CF lung, bacterial density increases and the microorganism switches to a mucoid form and to a stable biofilm mode of growth in which susceptibility to antimicrobials decreases. The high resistance of P.aeruginosa to multiple antimicrobials led to scenarios in which almost no treatment options are available. In this regard, the research on the introduction of less toxic antimicrobials as well as the use of pharmaceutical forms enabling dose reductions, longer administration intervals, and reduced systemic toxicity has been stimulated.

Therefore, the aim of this thesis was to develop nanoencapsulated colistin and tobramycin in lipid nanoparticles (SLN: Solid Lipid Nanoparticles and NLC: Nanostrucutured Lipid Carriers) and explore their antimicrobial activity versus free drug against P.aeruginosa clinical isolates from CF patients and to investigate the efficacy of these novel formulations in the eradication of biofilms, one of the most relevant mechanisms involved in persistence and in chronic infections.

ELABORATION AND CHARACTERIZATION

The main objective of the first part of this thesis was to elaborate and characterize lipid nanoparticles (SLN and NLC) as colistin and tobramycin carriers to treat P.aeruginosa lung infection.

The nanoparticles obtained displayed a 200–400 nm size, high drug encapsulation (79–94%) and a sustained drug release profile. The integrity of the nanoparticles was not affected by nebulization through a mesh vibrating nebulizer. Next, tobramycin-NLCs were able to overcome an artificial mucus barrier in the presence of mucolytic agents. Moreover, lipid nanoparticles loaded with both antimicrobials appeared to be less toxic than free drug in cell culture. Finally, an in vivo distribution experiment showed that nanoparticles spread homogenously through the lung and there was no migration of lipid nanoparticles to other organs, such as liver, spleen or kidneys.

STABILITY

The second essential point of this work concerns the stability of both types of lipid nanoparticles after freeze-drying.

The results showed that colistin-SLNs lost their antimicrobial activity at the third month; on the contrary, the antibacterial activity of colistin-NLCs was maintained throughout the study within an adequate range. In addition, colistin-NLCs exhibited suitable physic-chemical properties at 5 °C and 25 °C/60% relative humidity over one year. Altogether, colistin-NLCs proved to have better stability than colistin- SLNs.

The last part focuses on the study of the antimicrobial activity of SLN and NLC loaded with colistin and tobramycin against P.aeruginosa isolates from Sant Joan de Déu and Vall d’Hebrón hospitals CF patients.

Regarding the data documenting planktonic experiments, colistin nanoparticles had the same antimicrobial activity as free drug. The activity of tobramycin-loaded SLN was less than that of either tobramycin-loaded NLC or free tobramycin. However, in the relation to biofilms, nanoencapsulated antimicrobials were much more efficient than their free form. Moreover, the results showed the more rapid killing of P. aeruginosa bacterial biofilms by NLC-colistin than by free colistin. Nevertheless, the two formulations did not differ in terms of the final percentages of living and dead cells, which were higher in the inner than in the outer layers of the treated biofilms.

Since it seems clear than biofilms play a key role in respiratory infections in CF patients by P. aeruginosa, these formulations seem to us encouraging alternative to the currently available CF therapies.

P.aeruginosa és un dels principals patògens oportunistes colonitzadors del tracte respiratori dels pacients amb fibrosi quística (FQ) causant una infecció crònica. Una vegada aquest microorganisme ja està establert de manera crònica al pulmó, la densitat bacteriana augmenta i P.aeruginosa canvia de morfologia no mucosa a mucosa afavorint la formació de biofilm en el qual la susceptibilitat als antibiòtics es veu enormement disminuïda. L’elevada resistència de P.aeruginosa a múltiples antimicrobians ens condueix a un escenari on gairebé no hi ha opcions de tractament disponibles. En aquest sentit, la recerca en la introducció d’antimicrobians menys tòxics així com l’ús de noves formes farmacèutiques amb la capacitat de reduir la dosi, allargar els intervals d’administració així com reduir la toxicitat adquireix molta rellevància.

Per tant, l’objectiu d’aquesta tesi va ser desenvolupar nanopartícules lipídiques (Solid Lipid Nanoparticles: SLN y Nanostructured Lipid Carriers: NLC) carregades amb colistina I també les partícules amb tobramicina, explorar la seva activitat antimicrobiana comparant-la amb la seva forma lliure contra soques clíniques de P.aeruginosa aïllades de pacients amb FQ, i investigar l’eficàcia d’aquestes noves nanoformulacions en l’eradicació dels biofilms ja que és un dels mecanismes més rellevants associat a les infeccions cròniques.