(M1230-02-11) Formulation and Biological Evaluation of Spray Dried Microparticles Loaded with LL37 Antimicrobial Peptide

Purpose: The cathelicidin LL37 is studied as cationic antimicrobial peptide against drug resistant Pseudomonas aeruginosa (PA) infections especially occurring in the lungs of patients affected by cystic fibrosis (CF). However, its application is strongly limited by fast enzymatic degradation in vivo and low bioavailability. This calls the need of designed formulations able to deliver LL37 directly to the airways. The study reports the encapsulation of LL37 into spray dried microparticles (MPs) and the in vitro evaluation of the biological activity of LL37 and loaded MPs on PA strains. In addition, a preliminary hypothesis on how, from the structural point of view, LL37 might interact with bacterial membranes, considering a pore-forming mechanism, is presented.

Methods: Microparticles were prepared by spray drying an aqueous solution of LL37 at 15 % w/w together with trehalose at 75 % w/w and L-Leucine at 10 % w/w as the excipients forming the matrix of the microparticles. Spray drying parameters applied were an inlet temperature of 120°C, a feed rate of 3 ml/min, an air flow rate of 742 L/h, aspirator rate of 35 m³/h, and 0.7 mm nozzle. The powder obtained was characterized in terms of yield of drying, peptide content (%) by high performance liquid chromatography, microparticle’s size by laser diffraction and in vitro aerodynamic performance by means of a Next Generation Impactor (NGI) after the aerosolization from a RS01 DPI. The aerodynamic properties were expressed in terms of Emitted Fraction (EF%), Fine Particle Fraction (FPF%), and MMAD (um). In vitro biological evaluation was carried out with both pure peptide and loaded MPs on Pseudomonas aeruginosa PECHA 4 planktonic cells from a chronic wound clinical isolate. Bacterial growth inhibition was evaluated by Colony Forming Unit (CFU) assay and time killing assays. Circular dichroism studies were carried out to investigate the structural behaviour of LL37 against molecules capable of mimicking the composition of bacterial wall and membrane, i.e. lipopolysaccharide (LPS), detox lipopolysaccharide (LPS DETOX), and sodium dodecyl sulfate (SDS).

Results: The yield of the drying process was 58.06 ± 4.38 % with a peptide content of 89 % w/w indicating the successful formulation of LL37 into a dry powder. Laser diffraction measurements on MPs revealed a Dv₉₀ of 3.94 ± 0.02 um assuming the suitability for pulmonary administration of the powder produced, confirmed by the aerosolization studies. Indeed, the MMAD was 3.57 um, in accordance with the quality criteria required for inhalation powders for which the aerodynamic diameter must be between 2 and 5 um for lung deposition to occur. The emitted fraction (EF) value was 77 %, and the fine particle fraction (FPF) value was 67.19 %. LL37 shown a strong bacterial growth inhibition with an EC₅₀ of 0.346 uM and a PA killing after 8 minutes. This could be associated with a predominantly membranolytic action due to the ability of LL37 to form pores along the membrane because of electrostatic interaction with it. This interaction was simulated by circular dichroism studies where a conformation change of the peptide from random coil to 𝛂 helix was evidenced when in contact with 0.03 % LPS and SDS 50 mM. Once loaded in trehalose and leucine MPs LL37 shown a PA growth inhibition of 85%.

Conclusion: LL37 showed significant antimicrobial activity against Pseudomonas aeruginosa, presenting a promising minimal effective concentration that combined with an inhaled formulation capable of directing the peptide along the airways to the site of infection could represent a therapeutic alternative for the treatment of infections caused by drug-resistant bacteria.

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Time killing assay on Pseudomonas aeruginosa PECHA 4.

Circular dichroism spectra of LL37 dissolved in ultrapure water 100 uM and in contact and with LPS 0.001 %, LPS 0.01 %, LPS 0.03 %, and SDS 50mM.