Introduction Today, serious respiratory infections pose a global threat to the healthcare system. The problem is particularly acute in mycobacteriosis, which includes tuberculosis. The emergence of disease-resistant strains necessitates the development of novel medication systems. Most active compounds utilized in global tuberculosis treatment protocols have insufficient bioavailability in the lungs, requiring high-dose, long-term therapy. Bioavailability can be enhanced via biocompatible inhalation delivery techniques. The development of an inhalation system for the administration of anti-tuberculosis medications based on liposome complexes with different polymers appears to be a promising strategy. However, the interaction of the inhaled drug delivery systems with biological surfaces, such as the surface of the lungs coated with pulmonary surfactant, can play a critical role in achieving appropriate pharmacokinetic parameters and biodistribution. The goal of this research is to investigate the physicochemical patterns of interaction between the liposomal form of levofloxacin, functionalized with mannosylated chitosan (ChitMan), and bovine pulmonary surfactant.
Methods
Liposomal form of levofloxacin was obtained by routine passive loading technique. ChitMan coating was conducted by mixing with liposomes in base-molar ratio of 7:1 and incubated at RT for 30 min. ATR-FTIR spectroscopy was conducted by means of Bruker Tensor 27 machine, and ATR-FTIR microscopy was conducted by means of a Simex Mirkan-3 machine.
Results
Liposomal forms of levofloxacin were obtained based on anionic liposomes (DPPC - cardiolipin mass ratio 4:1) with a drug inclusion efficiency of 0.2 to 0.5 mg per 1 mg of lipids. The particles were characterized by a zeta potential of -22 mV and a hydrodynamic radius of 100 nm. The resulting complex was characterized by a zeta potential of +13 mV and a hydrodynamic radius 140 nm. Bovine lung surfactant was isolated using a classical extraction technique. According to IR microscopy data, the protein and lipid fractions are co-localized. The effect of liposomal forms of levofloxacin on the surface properties of a surfactant monolayer was analyzed using the Langmuir-Wilhelmy method. It was found that when an aliquot of the liposomal form of levofloxacin (LLEV) not coated with a polymer is added, the area per molecule – two-dimensional pressure curves – show the appearance of a region responsible for the fusion of the liposome membrane with the surfactant monolayer. In contrast, when adding an aliquot of polymer-coated vesicles (LLEV-Pol), stabilization of the surfactant is observed, but fusion does not occur. According to the AFM-microscopy, the interaction of surfactant with LLEV occurs over the entire surface area of the monolayer, while for LLEV-Pol binding is observed only at the surfactant-mica interface. To confirm the obtained data, a fluorescence microscopy study was carried out. The diffusion of the fluorescent label along the surfactant layer was monitored when liposomes or their complex with the polymer were applied. It was found that when free liposomes are added to the surfactant layer, the label is evenly distributed over the entire surface of the layer within an hour, while foci of fluorescence were observed for the liposome-polymer complex, indicating an obstacle to fusion.
Conclusions
In this work, we have demonstrated that ChiMan coating provides high adhesion to the pulmonary surfactant and prevents immediate fusion. This result indicates new strategies in inhaled drug delivery systems
Acknowledgements
The authors thank N. Melik-Nubarov for the fruitful discussions and the consultations during the experiments on the fluorescence microscopy.
Conflicts of interest
The authors declare that they have no conflict of interest in the publication of this article. The authors have no conflicts of interest to report in this work. Abstract was not submitted elsewhere and was first published here.
Funding
This study was supported by the Developmental program of Lomonosov MSU (ATR-FTIR spectrometer Bruker Tensor 27, ATR-FTIR microscope Simex Mikran-3).