Enzyme-Linked Lipid Nanocarriers for Coping Pseudomonal Pulmonary Infection. Would Nanocarriers Complement Biofilm Disruption or Pave Its Road?

Noha Nafee,1,2 Dina M Gaber,3 Alaa Abouelfetouh,4,5 Mustafa Alseqely,4 Martin Empting,6 Marc Schneider7 1Department of Pharmaceutics, College of Pharmacy, Kuwait University, Safat, 13110, Kuwait; 2Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt; 3Department of Pharmaceutics, Division of Pharmaceutical Sciences, College of Pharmacy, Arab Academy for Science, Technology and Maritime Transport, Alexandria, 1029, Egypt; 4Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt; 5Department of Microbiology and Immunology, Faculty of Pharmacy, Alamein International University, Alamein, 5060335, Egypt; 6Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Department of Antiviral and Antivirulence Drugs (AVID), Saarland University, Saarbrücken, 66123, Germany; 7Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, 66123, GermanyCorrespondence: Noha Nafee, Department of Pharmaceutics, College of Pharmacy, Kuwait University, Safat, 13110, Kuwait, Tel +96524634914 ; +96560300583, Fax +96524636843, Email noha.nafee@ku.edu.kwIntroduction: Cystic fibrosis (CF) is associated with pulmonary Pseudomonas aeruginosa infections persistent to antibiotics.Methods: To eradicate pseudomonal biofilms, solid lipid nanoparticles (SLNs) loaded with quorum-sensing-inhibitor (QSI, disrupting bacterial crosstalk), coated with chitosan (CS, improving internalization) and immobilized with alginate lyase (AL, destroying alginate biofilms) were developed.Results: SLNs (140– 205 nm) showed prolonged release of QSI with no sign of acute toxicity to A549 and Calu-3 cells. The CS coating improved uptake, whereas immobilized-AL ensured > 1.5-fold higher uptake and doubled SLN diffusion across the artificial biofilm sputum model. Respirable microparticles comprising SLNs in carbohydrate matrix elicited aerodynamic diameters MMAD (3.54, 2.48 μm) and fine-particle-fraction FPF (65, 48%) for anionic and cationic SLNs, respectively. The antimicrobial and/or antibiofilm activity of SLNs was explored in Pseudomonas aeruginosa reference mucoid/nonmucoid strains as well as clinical isolates. The full growth inhibition of planktonic bacteria was dependent on SLN type, concentration, growth medium, and strain. OD measurements and live/dead staining proved that anionic SLNs efficiently ceased biofilm formation and eradicated established biofilms, whereas cationic SLNs unexpectedly promoted biofilm progression. AL immobilization increased biofilm vulnerability; instead, CS coating increased biofilm formation confirmed by 3D-time lapse confocal imaging. Incubation of SLNs with mature biofilms of P. aeruginosa isolates increased biofilm density by an average of 1.5-fold. CLSM further confirmed the binding and uptake of the labeled SLNs in P. aeruginosa biofilms. Considerable uptake of CS-coated SLNs in non-mucoid strains could be observed presumably due to interaction of chitosan with LPS glycolipids in the outer cell membrane of P. aeruginosa.Conclusion: The biofilm-destructive potential of QSI/SLNs/AL inhalation is promising for site-specific biofilm-targeted interventional CF therapy. Nevertheless, the intrinsic/extrinsic fundamentals of nanocarrier–biofilm interactions require further investigation.Keywords: alginate lyase, chitosan, solid lipid nanoparticles, cystic fibrosis, Pseudomonas aeruginosa, quorum-sensing inhibitors