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

Abstract

Introduction

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

Acknowledgments

Authors acknowledge the Alexander von Humboldt Foundation, Germany for funding this research under The Research Group Linkage Programme.

Disclosure

Martin Empting reports a patent WO2020007938A1 pending to Helmholtz Centre for Infection Research (HZI), a patent EP20150104 pending to Helmholtz Centre for Infection Research (HZI), and a patent WO2021136805A1 pending to Helmholtz Centre for Infection Research (HZI). The authors report no other conflicts of interest in this work.