Smart lipid nanoparticles containing levofloxacin and DNase for lung delivery. Design and characterization

https://doi.org/10.1016/j.colsurfb.2016.03.040Get rights and content

Highlights

  • Levofloxacin is successfully encapsulated into lipid nanoparticles (SLN and NLCs).

  • NLC formulation exhibits a controlled release profile of Levofloxacin for 2 days.

  • The presence of DNase could decrease viscoelasticity found in the Cystic Fibrosis patient lungs.

  • The formulation shows an active antimicrobial activity against Cystic Fibrosis pathogens.

  • Lipidic nanoformulations are new alternatives for the Cystic Fibrosis treatment of infections.

Abstract

Levofloxacin (LV) is a hydrophilic broad-spectrum antibiotic commonly used in pulmonary treatment against recurrent infections of Pseudomonas aeruginosa, and particularly in cystic fibrosis (CF) disease. In order to study feasible carriers for LV, solid lipid nanoparticles (SLN) of myristyl myristate were prepared by the ultrasonication method in the presence of Pluronic®F68 under different experimental conditions and characterized by dynamic light scattering, optical, transmission and scanning electron microscopy for size and morphology. Alternatively, nanostructured lipid carriers (NLCs) were developed to improve LV encapsulation and storage. SLN showed 20.1 ± 1.4% LV encapsulation efficiency, while the NLCs encapsulated 55.9 ± 1.6% LV. NLC formulation exhibited a more controlled release profile than SLN formulation, but both showed a biphasic drug release pattern with burst release at the first 5 h and prolonged release afterwards, demonstrated by in vitro tests. The hydrodynamic average diameter and zeta potential of NLC were 182.6 ± 3.2 nm and −10.2 ± 0.2 mV, respectively, and were stable for at least 3 months. Additionally, DNase type I was incorporated into the formulations as a “smart” component, since the enzyme could help to decrease the viscoelasticity found in the lungs of CF patients and improves the antibiotic diffusion. FTIR, XRD, DSC, TGA and nitrogen adsorption isotherms of the nanoparticles indicate the presence of the loads in a noncrystalline state. The developed formulation showed an active antimicrobial activity against P. aeruginosa and even against other opportunistic pathogens such as Staphylococcus aureus. The presence of LV-loaded NLCs reduced the formation of a bacterial biofilm, which highlighted the significance of the nanodevice as a new alternative for CF treatment.

Graphical abstract

Lipid nanoparticles containing levofloxacin and DNase for lung delivery.

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Introduction

Among the illnesses with more cases of recurrent lung infections, cystic fibrosis (CF) is an inherited autosomal disease and considered one of the most common lethal genetic human disorders. It is produced by the defective function of a transmembrane conductance regulator protein, causing abnormalities in the airway physiology and mucociliary clearance [1]. These conditions are correlated with chronic lung infections recurrently caused by opportunistic pathogens, affecting more than 90% of all CF patients, and are the leading cause of morbidity and mortality [2]. Many bacterial species have been found in CF sputum, directly associated with lung disease, e.g., Burkholderia cepacia, Haemophilus influenza and Staphylococcus aureus, among others. However, Pseudomonas aeruginosa is considered the main cause of lethality [3]. The bacteria not only colonize lungs forming biofilms but could also mutate into mucoid-type strain, thereby producing considerable amounts of exopolysaccharide alginate [4]. These types of aggressive strains are capable of surviving strong antibiotic therapies and once established become extremely difficult to eradicate [5].

The progressive deterioration of lung functions in CF patients is also attributed to the airway obstruction caused by the accumulation of thick and purulent mucus. The dense secretions are mainly composed of mucus glycoproteins and DNA [6]. Although the origin of the extracellular DNA is not well established, it is suspected to be originated from necrotic neutrophils and lung tissues, and to a lesser extent, by the contribution of infecting bacteria [7]. In addition, the alginate synthesized by the bacteria will contribute to the viscoelasticity of the mucus and drastically worsen the patient́s health when the mucoid type of P. aeruginosa colonizes the lungs. As a result, an early treatment of exacerbations in pulmonary symptoms as well as effective antibiotic therapies become essential tools for increasing the life expectancy and life quality of patients [8], [9].

Aerosol delivery of antibiotics directly to the lungs has been proposed for the management of infections in CF patients [10]. This type of treatment increases the local concentration of the drug at the site of infection, thereby enhancing its antibacterial activity and reducing the selection of resistant species compared to the outcomes of systemic administration. Currently approved CF therapies involve the use of tobramycin or aztreonam lysine solutions as inhalation agents to treat P. aeruginosa infections [11], [12]. For several reasons, such as low drug efficacy, drug intolerance, novel emerging pathogens, and inconvenient dosing, there is a real need for alternative inhaled antimicrobial therapies to treat pulmonary infections caused by P. aeruginosa and other bacteria in CF patients [13].

Among the different vehicles for aerosol administration, solid lipid nanoparticles (SLN) have been developed in recent years as a potential system for lung delivery [14], [15], [16]. The selection of the lipid matrix is based on its properties of being nontoxic, biocompatible, of green chemical composition, physicochemical characteristics and small sizes that allow them to penetrate into almost all lung regions, enhancing the deep-lung deposition of the drugs and efficient biodistribution [17]. Many lipids were tested for the preparation of SLN, but myristyl myristate (MM) showed desirable properties as previously reported by Prof. Duran‘s group such as its ability to produce SLNs using simple hot high pressure homogenization [18]. Also, MM is considered as an excellent emulsion enhancer and effective thickening ester. Particularly, MM is 100% of natural origin (i.e., extracted from plants) and is present in some foods (i.e., it is considered safe). In addition, it was proved that MM showed no toxicity in oral acute and dermal toxicological tests on rats. Finally, the melting point of around 40 °C is desirable for the encapsulation of a wide range of molecules, even thermolabile drugs with biological activity.

An improvement in patients’ compliance due to the reduction of drug side effects in kidneys and extended drug dosing intervals due to the sustained drug release from SLNs was commonly observed [16]. In order to improve SLN properties, a new generation of nanoparticles named as nanostructured lipid carriers (NLC) has been developed. NLCs consist of lipid nanoparticles with liquid lipid included in their structure, which decreases the crystallinity degree of the matrix. After this modification, the storage stability, the encapsulation percentage of drugs and the release profiles can be improved [19].

In the present work, Levofloxacin (LV) has been selected as the antibiotic model for encapsulation, due to its potent activity against key pathogens in CF patients, including P. aeruginosa. Unlike tobramycin, LV activity is not reduced in the presence of mucus from CF sputum. Furthermore, LV has stronger antimicrobial activity than tobramycin and aztreonam in the presence of bacterial biofilms [20]. Previous reports indicate that aerosol administration of LV is more effective in terms of plasmatic concentrations and lower inhibitory concentration ratios in the airways than those obtained with parenteral or oral administration [21]. Although LV is one of the safety antibiotics among the quinolone family, its use in high concentrations to reach the therapeutic levels in the lungs results in serious nephrotoxicity side effects after oral administration [22]. The encapsulation of LV in effective therapeutic carriers for noninvasive systemic drug nanodelivery is an interesting alternative to be explored.

In the last years, LV encapsulation in SLN and NLCs has been mainly investigated for ocular delivery, but the feasibility of the carriers to transport the drug to the deepest sites of lungs is still unexplored [23]. The nanoparticles can offer a controlled release profile of the drug, prolonging the airway residence time in the lungs and preventing the emergence of resistant bacteria [24]. Moreover, lipid nanoparticles could prevent the crystallization of the free drug at the high administered concentrations, reducing the risks of LV-induced crystal nephropathy [25]. It is important to mention that the incorporation of an enzyme with the ability to break down the DNA chains will improve the antibiotic bioavailability [26], [27]. In this case, a DNase type I was incorporated into the formulation, and its effect on nanoparticle stability and activity was studied. The simultaneous presence of DNase and LV in the nanoparticles and the positive interaction between them are interesting properties that make the nanocarrier a “smart” system. DNase plays a mucolytic role in the microbial biofilm that surrounds the bacteria, enhancing the diffusion of the nanoparticles and consequently, the antimicrobial activity of fluoroquinolone. LV inhibits DNA gyrase and topoisomerase IV causing bacterial death and allowing DNase to hydrolyze all DNA material of the cells, including genomic and potential plasmids, avoiding the release of potential antibiotic resistance genes.

The aims of the present study are the development of lipid nanoparticles for an efficient encapsulation of LV in the presence of DNase as mucolytic enzyme, the characterization of the system in terms of the biophysicochemical properties (spectroscopies, microscopies, thermogravimetric and light scattering analysis) and the evaluation of the antimicrobial activity against common pathogens found in pulmonary infections.

Section snippets

Materials

The lipid myristyl myristate (Crodamol™ MM, melting point = 36–40 °C) and the oil (Crodamol™ GTCC-LQ, a fully saturated emollient triester, melting point = −5 °C) were kindly donated by Croda (Argentina). Levofloxacin (LV, (S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid), deoxyribonuclease (DNase) type I from bovine pancreas (MW≈ 31 kDa, isoelectric point≈ 5.2), DNase test agar with toluidine blue, Pluronic®F68 (cat # A-6973)

Preparation of SLN and NLC for levofloxacin encapsulation

Different SLN formulations were prepared by the ultrasonication method, and the encapsulation efficiency (EE) was calculated with Eq. (1) (Table 1). The SLN1 formulation was prepared by mixing the lipid phase with solid LV, followed by homogenization in 3.0% of Pluronic F68 (dissolved in distilled water). It showed a low EE with a value around 5%, possibly due to the tendency of LV molecules to migrate towards the aqueous solution. In the SLN2, the EE was increased more than twofold by

Conclusions

In the present work, novel solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLCs) containing LV were successfully prepared and characterized. A DNase was also incorporated into the formulations, and its relevant activity can be associated with a decrease in the mucus viscoelasticity found in the CF patients’ lungs, which improves the antibiotic diffusion. The preparation of nanoparticles can be tailored by changing the pH of the aqueous environment, the dissolution kinetics of

Acknowledgements

The present work was supported by Argentine grants from CONICET (National Council for Science and Technology, PIP 0498), The National Agency of Scientific and Technological Promotion (ANPCyT, PICT 2011-2116), Fundación Argentina de Nanotecnología, UNLP (National University of La Plata, 11/X545 and PRH 5.2). Dr. G.A. Islan thanks the “Grant for Young Researchers from UNLP 2014” and the “Program for academic mobility at teaching scale from AUGM” for financing some reagents used in the present

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      There is only one account that reports on the maximum of entrapped LV of ∼ 56 % that could be incorporated in NLCs (Islan et al., 2016). Another study loaded almost the same amount of LV on solid lipid nanoparticles (SLN) as that reported in the Islan et al. study (Islan et al., 2016), however, SLNs are known as not an optimum option for an entrapped drug in terms of the long term stability (Abdel Hady et al., 2020). Polymeric nanoparticles were the most chosen carriers to deliver LV, including the PLGA-based systems.

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