Liposomal antimicrobial agent formulations for the treatment of pulmonary infection in cystic fibrosis patients.

摘要

Cystic Fibrosis (CF) is a genetic recessive disease that conceivably passes through generations. The trait is recessive and thus the disease manifests itself only when two of the mutant CFTR alleles are present. CF is a childhood disease that affects many organs, but the most life threatening is associated with lung infections by Gram-negative Pseudomonas aeruginosa, and Burkholderia cepacia. Microbial infections are due to ion channel dysfunctions in the lung epithelial cells which create a favourable environment for bacterial colonization. Pathogen colonization of the lungs and development of resistance to antibiotics are a major concern to CF patients. Although several mechanisms account for antibiotic resistance, the most common mechanisms are the alteration of bacterial membrane permeability and the formation of biofilm communities.;Second, the antimicrobial efficacy of the co-encapsulation bismuth-ethanedithiol (BiEDT) and tobramycin in liposomal formulation were examined The combination of BiEDT with tobramycin in liposomes (LipoBiEDT-TOB) increased tobramycin efficacy and reduced BiEDT toxicity. I validated the LipoBiEDT-TOB formulation by examining its stability in different biological conditions and determining MICs and minimal bactericidal concentrations (MBCs). In addition, I generated a toxicity profile and determined the influence of LipoBiEDT-TOB formulation on bacterial adhesion to human lung cells in vitro. I found that LipoBiEDT-TOB exhibited lower MICs and eradicated highly resistant clinical strains of B. cenocepacia and P. aeruginosa. LipoBiEDT-TOB was significantly less toxic when compared to the free BiEDT, as assessed by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and lactate dehydrogenase (LDH) assay. Furthermore, the LipoBiEDT-TOB formulation suppressed bacterial adhesion to A549 lung cells.;Since bacterial virulence and biofilm formation in CF lungs are a consequence of bacterial communication or quorum sensing (QS), I also examined the effect of LipoBiEDT-TOB on QS molecule N-acyl homoserine lactone (AHL) secretion by P. aeruginosa isolates and its activity against biofilm-forming bacteria. In addition, CF sputum penetration by LipoBiEDT-TOB formulation was studied in order to determine whether our formulation is suitable for use in vivo where sputum are surrounding the infection site. The data indicated that LipoBiEDT-TOB significantly prevented QS molecule production and eradicated biofilm-forming bacteria at low concentrations. The formulation was also able to penetrate CF sputum as well; thus, tobramycin efficacy was enhanced.;Finally, I established a strategy by co-encapsulating gallium with gentamicin in a liposomal formulation (Lipo-Ga-GEN). I proved that the product can enhance gentamicin efficacy and reduce gallium toxicity. This characterization included studying the stability of the antibiotics within the formulation in different biological conditions, MICs, MBCs, minimum biofilm eradication concentration (MBEC), QS molecules reduction, and gallium toxicity profile. The formulation effectively suppressed a highly resistant planktonic strain of P. aeruginosa, completely eradicated the biofilm bacteria, and blocked the QS molecules at very low concentrations. In addition, gallium delivered to lung cells as Lipo-Ga-GEN formulation was less toxic to lung epithelial cells as indicated by an in vitro cell viability assay.;I have developed liposomal antimicrobial agent formulations which reduce the toxicity and increase the delivery of the agent to the pathogens. These liposomal formulations contained aminoglycosides either alone or in combination with bismuth or gallium. First, I developed different liposomal antimicrobial formulations consisting of amikacin, gentamicin, or tobramycin. Then I examined their efficacy against pathogenic planktonic bacteria of clinical isolates of B. cenocepacia. This included studying the stability of the liposomal-antibiotics formulations in different biological conditions, the encapsulation efficiencies of the drugs, liposomal interaction and penetration of bacterial cell membranes. Data indicate favorable encapsulation efficiencies for amikacin, gentamicin or tobramycin and tolerable stability in biological conditions such as bronchoalveolar lavage fluid (BAL) and plasma at thirty-seven degrees celsius. The transmission electron microscopy (TEM) studies along with lipid-mixing assays and fluorescence-activated cell-sorting (FACS) analysis revealed close interaction between liposomal bilayers and bacterial cell membranes of B. cenocepacia. In addition, liposomal formulations enhanced the antibiotics' bactericidal activity by reducing minimal inhibitory concentrations (MICs) of highly antibiotic-resistant strains of B. cenocepacia.;In conclusion, all the liposomal formulations examined enhance the antimicrobial efficacy of the drugs through altering bacterial membrane permeability in vitro. These data also suggest that co-encapsulation of aminoglycosides with bismuth or gallium in liposomes enhances their efficacy and reduces their toxicity. In addition, the formulations prevent the production of QS molecules which might playa role in enhancing the drug's antimicrobial efficacy on biofilm-forming bacteria.