Inhalable liposomal powder formulations for co-delivery of synergistic ciprofloxacin and colistin against multi-drug resistant gram-negative lung infections

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Abstract

The aim of this study was to design and characterize dry powder inhaler formulations of ciprofloxacin and colistin co-loaded liposomes prepared by the ultrasonic spray-freeze-drying (USFD) technique. Liposomal formulations and powder production parameters were optimized to achieve optimal characteristics and in-vitro performance such as encapsulation efficiency (EE), particle size, particle distribution index (PDI), fine particle fraction (FPF), emitted dose (ED) and in vitro antibacterial activity. The formulation (F6) with the mannitol (5% w/v) as the internal lyoprotectant and sucrose (5%, w/v), mannitol (10%, w/v) and leucine (5%, w/w) as the external lyoprotectants/aerosolization enhancers showed an optimal rehydrated EE values of ciprofloxacin and colistin (44.9 ± 0.9% and 47.0 ± 0.6%, respectively) as well as satisfactory aerosol performance (FPF: 45.8 ± 2.2% and 43.6 ± 1.6%, respectively; ED: 97.0 ± 0.5% and 95.0 ± 0.6%, respectively). For the blank liposomes, there was almost no inhibitory effect on the cell proliferation in human lung epithelial A549 cells, showing that the lipid materials used in the liposome formulation is safe for use in pulmonary drug delivery. The cytotoxicity study demonstrated that the optimized liposomal formulation (F6) was not cytotoxic at least at the drug concentrations of colistin 5 μg/mL and ciprofloxacin 20 μg/mL. Colistin (2 mg/L) monotherapy showed no antibacterial effect against P. aeruginosa H131300444 and H133880624. Ciprofloxacin (8 mg/L) monotherapy showed moderate bacterial killing for both clinical isolates; however, regrowth was observed in 6 h for P. aeruginosa H133880624. The liposomal formulation displayed superior antibacterial activity against clinical isolates of Pseudomonas aeruginosa H131300444 and P. aeruginosa H133880624 compared to each antibiotic per se. These results demonstrate that the liposomal powder formulation prepared by USFD could potentially be a pulmonary delivery system for antibiotic combination to treat multi-drug resistant Gram-negative lung infections.

Introduction

The pulmonary infections caused by multidrug-resistant (MDR) Gram-negative bacteria, such as Pseudomonas aeruginosa are a major problem in the global healthcare system (Aliyu et al., 2017, Hawkey et al., 2018). Unfortunately, over the past a few decades the development of new antibiotics against these Gram-negative ‘superbugs’ had lagged far behind the emergence spread of antibiotic resistant pathogens (Li et al., 2006). Antibiotic combination therapies have been widely recognized as an effective strategy for the treatment of MDR Gram-negative infections where monotherapy has failed (Suzuki et al., 2017, Tamma et al., 2012).

Ciprofloxacin (Cipro; Fig. S1a) is a broad-spectrum antibiotic belonging to the quinolone family. The primary mode of action of ciprofloxacin involves inhibition of the bacterial DNA gyrase and topoisomerase IV (Lim et al., 2018, Rehman et al., 2019). Colistin (Col, also known as polymyxin E; Fig. S1b) is a cationic lipopeptide antibiotic composed of a cyclic decapeptide and a fatty acyl chain (Barker et al., 2017, Kwa et al., 2007, Liu et al., 2016). The amphipathic structure of colistin facilitates its interaction with the bacterial membrane, self-promoted insertion and disruption of the membrane, which culminates into bacterial cell death (Moffatt et al., 2010, Wang et al., 2018a, Yu et al., 2015). Intravenous colistin is not very effective against lung infections because only a very small proportion of the administrated dose reaches the lung tissue from the systemic circulation (Garnacho-Montero et al., 2003, Lin et al., 2018a, Lin et al., 2018b). Simply, increasing the intravenous dose is not an option due to dose-limiting neuro- and nephron-toxicity of polymyxins (Dai et al., 2018, Stichtenoth et al., 2017). One alternative is to deliver synergistic antibiotics such as ciprofloxacin and colistin directly to the respiratory tract, thereby achieving high drug concentrations directly at the infection sites and minimizing systemic toxicity (Brillault et al., 2017, Liu et al., 2015, Zhou et al., 2015, Mangal et al., 2019). Ciprofloxacin in combination with colistin has been shown to display synergistic antimicrobial activity and minimize the emergence of resistance against lung infections caused by P. aeruginosa in cystic fibrosis patients (Hoiby et al., 2005). Combination of oral therapy of ciprofloxacin and parenteral colistin against bacterial infection in neutropenic patients has been shown to prevent emergence of ciprofloxacin resistance over periods as long as 10 years (Prentice et al., 2001).

Liposomes hold great promise in the realm of drug delivery, and have been successfully developed into several FDA approved pharmaceutical products (Crommelin and Storm, 2003, Manconi et al., 2017, Torchilin, 2005, Wong et al., 2003, Zununi Vahed et al., 2017). Notably, recent studies have highlighted the advantages of liposomes for pulmonary drug delivery, such as biocompatibility, ability to load both hydrophilic and hydrophobic agents, prolonging drug retention time, reduced drug toxicity and minimized pulmonary clearance (Castoldi et al., 2017, Colzi et al., 2015, Manca et al., 2014, Wardlow et al., 2016). Liposomes may allow controlled drug release and thus reduce the dosing frequency, mask the taste of bitter compounds and reduce the pulmonary irritation associated with the drug (Chai et al., 2019, Cipolla et al., 2016, Wang et al., 2018b). Importantly, liposomes can facilitate targeting of specific pulmonary cells, such as macrophages that harbor bacteria (Cipolla et al., 2013).

Amongst the different pulmonary drug delivery systems, dry powder inhaler formulations (DPIs) are widely accepted because of the superior chemical stability of drugs in their solid form and the convenience for patients to carry and use the delivery devices, as compared to delivery via nebulization (Frijlink and De Boer, 2004, Garcia-Contreras and Smyth, 2005, Telko and Hickey, 2005). Formulating liposomes into dry powders is attractive for pulmonary administration, particularly with respect to the ability to control delivery, reduce toxicity, free of propellant, improved patient compliance and satisfactory chemical stability (Chennakesavulu et al., 2018, Chougule et al., 2008, Vermehren et al., 2006). Ultrasonic spray-freeze-drying (USFD) has been applied for the production of DPI liposomal formulations as this method can generate low-density porous particles with improved aerosolization performance (D'Addio et al., 2013, Gao et al., 2011, Ye et al., 2017). In the USFD process, the liquid is atomized into droplets by the ultrasonic nozzle (Rogers et al., 2002). Then the droplets are sprayed directly into the liquid nitrogen which rapidly turns the atomized droplets into frozen spheres (Yin et al., 2014). Followed by freeze-drying, porous particles with a low density have relatively lower aerodynamic diameters (D'Addio et al., 2013, Edwards et al., 1997).

Our present study aimed to apply the USFD technique to develop inhalable liposomal DPI formulations containing synergistic ciprofloxacin and colistin. The morphology (by cryo-TEM) and the cytotoxicity of the ciprofloxacin-colistin-loaded liposomal (Cipro-Col-Lips) suspensions were studied. The effect of different lyoprotectants including sucrose, leucine, and mannitol on powder in vitro aerosolization and the encapsulation efficiency of the reconstituted formulations were determined. Physicochemical properties (i.e. particle size, particle density, and hygroscopicity), morphology, X-ray diffraction patterns of the dry powders were also evaluated. Outcomes of the present study showed that the liposomal powder formulation could potentially be a viable pulmonary delivery system for treating multi-drug resistant Gram-negative lung infections.

Section snippets

Materials

Ciprofloxacin hydrochloride monohydrate (ciprofloxacin or Cipro) and Colistin sulfate (colistin or Col) were purchased from BetaPharma Co., Ltd. (Wujiang, JiangSu, China). Hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphoglycerol sodium salt (DSPG-Na, abbreviated as DSPG in the text) and N-(methylpolyyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (DSPE-PEG-OMe, abbreviated as PEG in the text) were purchased from NOF

Characterization of Cipro-Col-Lips suspension before USFD

EE, particle size and zeta potential of the Cipro-Col-Lips suspensions before USFD were shown in Table 2. The results showed that different hydration media had no effect on the EE of ciprofloxacin (all above 90%) and colistin (all above 40%). The mean particle sizes of different Cipro-Col-Lips suspension were all under 155 nm and PDI were <0.25.

Morphology of liposomal formulations were visualized by cryo-TEM. As shown in Fig. 2, the liposomes displayed spherical shape with a visible lipid

Conclusions

In the present study, we developed ciprofloxacin and colistin liposomal DPI formulations via ultrasonic spray-freeze-drying with satisfactory aerosol performance (ED > 95% and FPF ~ 50%). Cryo-TEM imaging revealed that the liposomal Cipro-Col-Lips DPI formulations displayed a spherical shape with a discernable lipid membrane and porous structure. The rehydrated EEs of ciprofloxacin and colistin from the optimized Cipro-Col-Lips formulation (F6) were 44.9 ± 0.9% and 47.0 ± 0.6%, respectively.

CRediT authorship contribution statement

Shihui Yu: Data curation, Formal analysis, Writing - original draft. Shaoning Wang: Conceptualization, Formal analysis, Writing - review & editing. Peizhi Zou: Data curation. Guihong Chai: Supervision, Validation. Yu-Wei Lin: Data curation, Formal analysis. Tony Velkov: Conceptualization, Writing - review & editing. Jian Li: Conceptualization, Funding acquisition, Supervision, Writing - review & editing. Weisan Pan: Conceptualization, Supervision, Writing - review & editing. Qi Tony Zhou:

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.

Acknowledgements

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institute of Health under Award Number R01AI146160. Qi Tony Zhou and Jian Li are also supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01AI132681. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of

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