Ciprofloxacin-loaded PLGA nanoparticles against cystic fibrosis P. aeruginosa lung infections

Abstract

Current pulmonary treatments against Pseudomonas aeruginosa infections in cystic fibrosis (CF) lung suffer from deactivation of the drug and immobilization in thick and viscous biofilm/mucus blend, along with the general antibiotic resistance. Administration of nanoparticles (NPs) with high antibiotic load capable of penetrating the tight mesh of biofilm/mucus can be an advent to overcome the treatment bottlenecks. Biodegradable and biocompatible polymer nanoparticles efficiently loaded with ciprofloxacin complex offer a solution for emerging treatment strategies. NPs were prepared under controlled conditions by utilizing MicroJet Reactor (MJR) to yield a particle size of 190.4 ± 28.6 nm with 0.089 PDI. Encapsulation efficiency of the drug was 79% resulting in a loading of 14%. Release was determined to be controlled and medium-independent in PBS, PBS + 0.2% Tween 80 and simulated lung fluid. Cytotoxicity assays with Calu-3 cells and CF bronchial epithelial cells (CFBE41o⁻) indicated that complex-loaded PLGA NPs were non-toxic at concentrations ≫ MIC_cipro against lab strains of the bacteria. Antibacterial activity tests revealed enhanced activity when applied as nanoparticles. NPs’ colloidal stability in mucus was proven. Notably, a decrease in mucus turbidity was observed upon incubation with NPs. Herewith, ciprofloxacin complex-loaded PLGA NPs are introduced as promising pulmonary nano drug delivery systems against P. aeruginosa infections in CF lung.

Introduction

Chronic pulmonary infections, among which Pseudomonas aeruginosa is known to be the major pathogen, are reported to be the main cause of mortality among cystic fibrosis (CF) patients [1]. In addition to intravenous (i.v.) or oral administration [2], [3], during the last decades, repeated courses of high doses of nebulized and inhaled antibiotics have been applied extensively for treatment of early infections as a preventive action against the mucoidic bacteria [2], [3], [4], [5], [6]. However, once infection is established in the airways, it is almost impossible to eradicate it. The established biofilm anchors the bacteria to their environment, protects the bacteria and prevents drugs reaching minimum inhibition concentration (MIC) at the site of action to kill bacteria [7]. Biofilm-forming mucoid strains of P. aeruginosa are less susceptible to antibiotics than their planktonic counterparts as a result of different resistance mechanisms. Bacteria show slow growth or proliferation patterns or switch to anaerobic state at certain regions of the biofilm [8], thus bacterial biofilms show heterogenous composition [9]. In addition, diffusion rates of antibiotics in the biofilm are lower in comparison to water, they are prone to inactivation by the biofilm components, and penetration of the antibiotics is strongly affected by the properties of both antibiotics and the heterogeneous biofilm matrix [10], [11], [12], [13]. Consequently, antibiotic resistance is enhanced as a result of long term exposure. Under such disease conditions, a local nano drug delivery system capable of penetrating the thick mucus and biofilm, releasing antibiotic in a controlled manner at the site of action can be an intriguing approach to overcome treatment bottlenecks [14], [15], [16]. Particles should be engineered to possess particle size smaller than the CF mucus/biofilm meshes [17], [10], [18], and to prevent adsorption only onto the biofilm and entrapment in the biofilm via surface properties [19], [20], [21]. With this perspective, the development of pulmonary drug delivery systems for improved interactions with local environment is needed [22].

Pulmonary drug delivery offers many advantages including avoiding first pass effect, reduced systemic side effects, delivering higher doses at the site of action thus increased local concentration [23], higher bioavailability [24], increasing patient compliance and being a non-invasive drug delivery method [25]. Controlled particle size is one of the prerequisites of pulmonary drug delivery and lung deposition.

Engineering nanoparticles at particle size level requires full control over the preparation method. Traditional nanoparticle precipitation techniques lack control over nucleation and growth process, thus particle size and polydispersity index (PDI). Reproducibility problems at lab scale and scale-up problems at industrial level are faced during early and later stages of the development phase. Since particle size and distribution is a critical quality attribute for development of pulmonary nanomedicine against P. aeruginosa infections in CF, MicroJet Reactor (MJR) technology, a precise preparation technique performed under controlled conditions, was employed. MJR enables control over the whole process parameters and environment, and turbulent like mixing in micron-volume caused by impinging jets provides high nanoparticle quality. NP quality can be theoretically defined as a function of parameters that govern the whole process [26].

Biocompatibility and biodegradability of the nano-carrier play an important role for pulmonary nanomedicines, especially for the disease cases like CF, where mucociliary clearance (MCC) is impaired [27], [28]. Thus, possible accumulation of the carrier is more likely. Poly(lactic-co-glycolic) acid (PLGA) applications have attracted attention following the first FDA approved product and have been successfully used since decades for micro-formulated drug delivery systems [29], [30], [31], [32], [33]. In addition to its biocompatibility hence low toxicity, it also offers predictable biodegradation kinetics [32]. Even though PLGA is not approved for pulmonary applications, yet, these characteristics make it a promising excipient for a clinically applicable formulation. Ciprofloxacin, a fluoroquinolone antibiotic, which is widely used for treatment of P. aeruginosa lung infections in CF patients, was chosen as model drug to be encapsulated with high drug loading in PLGA nano-carriers. Encapsulation of active pharmaceutical ingredients such as ciprofloxacin, showing pH dependent solubility and limited organic solvent solubility, compromise high drug loading regardless of preparation technique. In order to achieve potentially clinically effective drug loading, counter-ion complex of ciprofloxacin was employed [34]. Nanoformulations’ success also depends on their potential to reach the bacteria. Thus interaction with mucus, dissolution profile to sustain the local concentration at the site of action and release kinetics to understand the underlying physical and chemical phenomena were characterized. Additionally, safety and efficacy of the nanoformulation have been evaluated to ensure sustainability of the developed nanomedicine.