Colistin-loaded aerosolizable particles for the treatment of bacterial respiratory infections

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Abstract

Compared to parenteral administration of colistin, its direct pulmonary administration can maximize lung drug deposition while reducing systemic adverse side effects and derived nephrotoxicity. Current pulmonary administration of colistin is carried out by the aerosolization of a prodrug, colistin methanesulfonate (CMS), which must be hydrolized to colistin in the lung to produce its bactericidal effect. However, this conversion is slow relative to the rate of absorption of CMS, and thus only 1.4 % (w/w) of the CMS dose is converted to colistin in the lungs of patients receiving inhaled CMS. We synthesized several aerosolizable nanoparticle carriers loaded with colistin using different techniques and selected particles with sufficient drug loading and adequate aerodynamic behavior to efficiently deliver colistin to the entire lung. Specifically, we carried out (i) the encapsulation of colistin by single emulsion-solvent evaporation with immiscible solvents using polylactic-co-glycolic (PLGA) nanoparticles; (ii) its encapsulation using nanoprecipitation with miscible solvents using poly(lactide-co-glycolide)-block-poly(ethylene glycol) as encapsulating matrix; (iii) colistin nanoprecipitation using the antisolvent precipitation method and its subsequent encapsulation within PLGA nanoparticles; and (iv) colistin encapsulation within PLGA-based microparticles using electrospraying. Nanoprecipitation of pure colistin using antisolvent precipitation showed the highest drug loading (55.0 ± 4.8 wt%) and spontaneously formed aggregates with adequate aerodynamic diameter (between 3 and 5 μm) to potentially reach the entire lung. These nanoparticles were able to completely eradicate Pseudomonas aeruginosa in an in vitro lung biofilm model at 10 µg/mL (MBC). This formulation could be a promising alternative for the treatment of pulmonary infections improving lung deposition and, therefore, the efficacy of aerosolized antibiotics.

Introduction

Colistin (polymyxin E) is a polypeptide antibiotic of last resort, which is normally used in combination with other antibiotics in the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative infections (Ahn et al., 2017). Colistin binds to lipid A of the outer membrane lipopolysaccharides (OM) of Gram-negative bacteria, then disrupts the OM and inner membrane and causes cell lysis. To reduce its neuro- and nephrotoxicity, colistin is administered as the prodrug colistin sodium methanesulfonate (CMS), which is hydrolyzed after parenteral administration or after inhalation releasing its active form (i.e., colistin). However, intravenous (IV) colistin shows limited pulmonary diffusion and has significant nephrotoxicity (Rello et al., 2017). Nowadays, CMS is administered by the pulmonary route as a powder, in PEG-gelatin capsules (Colobreathe®), or as solutions using nebulizers (eFlow® rapid, PARI LC PLUS®), to treat chronic pulmonary infections caused by Pseudomonas Aeruginosa in patients with cystic fibrosis (Schuster et al., 2013).

However, CMS is not an optimal prodrug. In fact, CMS is inactive against P. aeruginosa and has to be converted by non-enzymatic hydrolysis into colistin to produce its bactericidal effect (Bergen et al., 2006). This conversion is slow compared to the CMS pulmonary absorption rate (Boisson et al., 2017, Boisson et al., 2014, Couet et al., 2012). In addition, in critically ill patients receiving CMS by nebulization, only 1.4 % (w/w) of the CMS dose is converted into colistin in the lung (Boisson et al., 2014). Therefore, relatively high CMS concentrations are required to obtain effective colistin concentrations in the pulmonary epithelial lining fluid (ELF). Still, the International Consensus Guidelines for the Optimal Use of the Polymyxins, endorsed by the American College of Clinical Pharmacy, the European Society of Clinical Microbiology and Infectious Diseases, the Infectious Diseases Society of America, the International Society for anti-infective Pharmacology, the Society of Critical Care Medicine, and the Society of Infectious Diseases Pharmacists indicate that despite the risks associated to the use of nebulized colistin its potential benefits outweigh them due to a reduced associated nephrotoxicity and increased concentration of the drug in the lung parenchyma; although those professional associations indicate that more randomized clinical trials are needed to draw solid conclusions (Tsuji et al., 2019). Therefore, site-specific drug delivery systems for the efficient pulmonary delivery of colistin can increase the antibiotic local concentration while avoiding unwanted side effects making them adequate even for patients with other underlying pulmonary conditions.

The “respirable mass” (mass that is deposited on the lung parenchyma by gravitational sedimentation) is maximum for particles having a mass median aerodynamic diameter between 1 and 5 μm. Larger particles are retained by inertial impaction or interception in the upper airways (i.e., oropharyngeal deposition) and smaller particles remain suspended for prolonged periods of time by diffusion being a large fraction exhaled. Alveolar deposition is then maximum for those particle sizes in the 1 to 5 μm range and also for submicron particles (<100 nm) (Kodros et al., 2018) but it is complex to maintain a stable aerosol having submicron sizes, mainly because of their high surface reactivity and tendency to agglomeration.

Different strategies have been followed in order to maximize drug accumulation in the lung parenchyma when treating respiratory infections. Drug particle sizes have been tuned in order to maximize deposition and avoiding exhalation in this targeted micron size (Miller et al., 2021). Also, thiolated drug delivery vehicles (e.g., polymers), commonly named thiomers, have been extensively used in pulmonary drug delivery to promote antibiotic-loaded carrier mucoadhesion while avoiding mucus turn over due to their ability to form disulfide bonds with the cysteine-rich subdomains of lung mucus glycoproteins via thiol-disulfide exchange reactions (Dünnhaupt et al., 2015). Hydrophobic interactions between most of the hydrophobic polymers used are also responsible for the observed mucoadhesion (Nafee et al., 2014). PEGylation is also a common strategy to either promote mucoadhesion or mucus-penetrating ability depending on its structure (i.e., linear, brush) and molecular weight, avoiding, at the same time, mucociliary clearance. PEG mucoadhesion ability has been attributed to the hydrogen bonding of the two lone electron pairs of oxygen in PEG repeat unit with mucin (Guichard et al., 2017). Its mucus-penetrating ability has been attributed to the neutral hydrophilic character and steric hindrance of high-density low molecular weight PEGs functionalizing drug-loaded nanoparticles which avoid particle agglomeration or irreversible aggregation. PEGylation on polymer nanoparticles improves lung retention and drug availability due to its rapid diffusion through the mucus layer reaching the underlying epithelial cells (Schneider et al., 2017).

The aliphatic polyester polylactic-co-glycolic acid (PLGA) is one of the most frequently used polymers for the sustained delivery of antibiotics due to its hydrolytic nature which makes it to decompose in biodegradable endogenous lactic and glycolic acids. Its molecular weight and the molar ratio of the monomers present can be tuned to provide with different degradation half-lives and the consequent specific release kinetics. Aerosolizable colistin has been previously encapsulated within PLGA showing prolonged antibiofilm action against biofilm-forming P. aeruginosa ATCC 27853 with just one single application compared to the multiple treatments required to exert the same antimicrobial action when using the free antimicrobial peptide (d’Angelo et al., 2015b). However, colistin loadings achieved using this single emulsion solvent evaporation synthesis technique were not very high (i.e., 1.27 wt%) (d’Angelo et al., 2015b). Other works using a similar approach for the formation of colistin loaded PLGA microparticles also rendered moderate drug loadings <5 wt% (Nanjo et al., 2013). Using double emulsion solvent evaporation Shi et al. (Shi et al., 2010) encapsulated colistin sulfate salt in PLGA microparticles having drug loadings up to 16 wt%. Despite all these efforts, it has not yet been possible to obtain high drug loadings of more than 50 wt% in PLGA micro- and nanoparticles with hydrodynamic sizes suitable for pulmonary delivery. Herein, we propose the encapsulation of colistin in PLGA nanoparticles intended for pulmonary delivery using: (i) single emulsion-solvent evaporation; (ii) nanopreciptation using miscible solvents with PLGA-PEG, poly(lactide-co-glycolide)-block-poly(ethylene glycol), as encapsulating matrix; (iii) colistin nanoprecipitation using the antisolvent precipitation method to render nanoparticles used either standing-alone or encapsulated within PLGA nanoparticles; and (iv) colistin encapsulation within PLGA-based microparticles using electrospraying.

Section snippets

Materials and methods

Hydroxypropyl methylcellulose (HPMC, Mn ∼ 10,000), poly(ethylene glycol) (PEG300, Mn 300), Pluronic® F68 (average Mw 8350 Da), poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-PLGA, PEG average Mn 5,000, PLGA Mn 25,000, lactide:glycolide 50:50), sodium hydroxide (≥98 %), the contrast agent phosphotungstic acid (reagent grade), as well as N,N-dimethylformamide (DMF) and ethyl acetate, used as solvents, were supplied by Sigma-Aldrich. Acros Organics provided the colistin

Preparation and characterization of colistin-loaded nano and microparticles

In this work, several different polymeric nano- and microparticles have been developed for the encapsulation of colistin. Depending on the particle size desired, and in order to achieve high drug loadings, different fabrication techniques were used to obtain the desired colistin-loaded particles; namely, o/w emulsification, nanoprecipitation, complex coacervation, and electrospraying.

The oil-in-water (o/w) single emulsion solvent evaporation method was successfully used for preparing PLGA-col

Discussion

Pulmonary delivery of antibiotic-loaded nanoparticles has a clear advantage over enteral delivery because first-pass metabolism is avoided and because by adjusting the size and surface characteristics of the particles, it is possible to target the drug to the lung parenchyma, providing localized treatment of pulmonary infections. Here we evaluated different protocols for the synthesis of colistin nanoparticles having high drug loadings and an appropriated particle size for pulmonary delivery.

Conclusions

Out of four different synthesis protocols tested to obtain high colistin loadings and appropriated aerodynamic sizes to efficiently reach the lung parenchyma, the nanocoacervation of colistin using the antisolvent precipitation method outperforms the other ones (i.e., single emulsion solvent evaporation, nanoprecipitation and electrospraying). The nanocoacervation of the unprotonated form of colistin within HPMC allows reaching drug loadings as high as 55.0 wt% ± 5.0 showing a mass median

CRediT authorship contribution statement

Guillermo Landa: Investigation, Methodology, Writing – original draft, Visualization. Teresa Alejo: Investigation, Writing – original draft. Theo Sauzet: Investigation, Methodology. Julian Laroche: Investigation, Methodology. Victor Sebastian: Formal analysis, Writing – review & editing, Visualization. Frederic Tewes: Conceptualization, Methodology, Resources, Writing – review & editing, Funding acquisition, Project administration. Manuel Arruebo: Conceptualization, Methodology, Resources,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Financial support from the Spanish Ministry of Science and Innovation (grant numbers PID2020-113987RB-I00, CTQ2017-84473-R and PID2021-127847OB-I00) is gratefully acknowledged. This manuscript is the result of the projects PDC2021-121405-I00 and PDC2022-133866-I00, founded by MCIN/AEI/10.13039/501100011033 and by the European Union “NextGenerationEU”/PRTR. The regional Governments of Aragon and Nouvelle-Aquitaine (by means of the Order PRE/2106/2017 and arrêté n°19000160 EMPIRE project) are

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