Mucoadhesive Rifampicin-Liposomes for the Treatment of Pulmonary Infection by Mycobacterium abscessus: Chitosan or ε-Poly-L-Lysine Decoration

Abstract

Mycobacterium abscessus (Mabs) is a dangerous non-tubercular mycobacterium responsible for severe pulmonary infections in immunologically vulnerable patients, due to its wide resistance to many different antibiotics which make its therapeutic management extremely difficult. Drug nanocarriers as liposomes may represent a promising delivery strategy against pulmonary Mabs infection, due to the possibility to be aerosolically administrated and to tune their properties in order to increase nebulization resistance and retainment of encapsulated drug. In fact, liposome surface can be modified by decoration with mucoadhesive polymers to enhance its stability, mucus penetration and prolong its residence time in the lung. The aim of this work is to employ Chitosan or ε-poly-L-lysine decoration for improving the properties of a novel liposomes composed by hydrogenated phosphatidyl-choline from soybean (HSPC) and anionic 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt (DPPG) able to entrap Rifampicin. A deep physicochemical characterization of polymer-decorated liposomes shows that both polymers improve mucoadhesion without affecting liposome features and Rifampicin entrapment efficiency. Therapeutic activity on Mabs-infected macrophages demonstrates an effective antibacterial effect of ε-poly-L-lysine liposomes with respect to chitosan-decorated ones. Altogether, these results suggest a possible use of ε-PLL liposomes to improve antibiotic delivery in the lung.

Keywords: Chitosan; Mycobacterium abscessus; Rifampicin; liposomes; mucoadhesion; polymer decoration; ε-poly-L-lysine.

Figure A1

Rifampicin release profile until up to 48 h from liposomes post aerosolization. Data were obtained as the mean of three independent experiments.

Figure A2

Hydrodynamic diameter (DH), and ζ-potential of decorated and undecorated liposomes in presence of mucin.

Figure A3

Antimicrobial effect of Chit-decorated RIF-liposomes. To evaluate intracellular bacterial growth, dTHP-1 were infected with Mabs for 3 h at 37 °C at a MOI of 10. After infection, extracellular bacilli were eliminated by 1 h incubation with 250 ug/mL amikacin. Subsequently, dTHP-1 cells were treated for 18 h with unloaded liposomes, free RIF, RIF-loaded liposomes, RIF-loaded Chit-decorated liposomes. Finally, macrophages were lysed with deoxycholate 1% and then samples were diluted in PBS-tween 80 and CFU quantified by plating bacilli in triplicate on 7H10. Replication index was calculated as the ratio between the CFU obtained 18 h of treatment and those obtained immediately after infection, before the addition of the stimuli. The results are shown as mean ± standard deviation of the values obtained from triplicate of each condition and are representative of three different independent experiments. n.s. = non-significant, *** p < 0.001 and **** p < 0.0001 by two-tailed Student’s t-test.

Figure 1

TEM images (PTA staining) of liposomal formulations, LipoRIF (A), LipoRIF-Chito (B), LipoRIF-ε-PLL (C) observed as prepared and after interaction with Mucin (D,E,F, respectively).

Figure 2

AFM topographical images (Height sensor channel) of liposomal formulations: LipoRIF (A), LipoRIF-Chito (B), LipoRIF-ε-PLL (C) observed as prepared and after interaction with mucin (D,E,F, respectively). The false color scale of height is shown on the left of each image. Insets in upper panels show the Prewitt-filtered images of the marked region.

Figure 3

(A–D) Result of investigation on physicochemical stability of decorated liposomes until up to 90 days at 4 °C and room temperature. (E) Stability studies over time of Rifampicin-loaded liposomes at 2 different storage temperatures over a 90-day period. (F) Effect of culture media at different incubation times on hydrodynamic diameter and ζ-potential of liposomes.

Figure 4

Rifampicin release profile until up to 48 h. Data were obtained as the mean of three independent experiments.

Figure 5

Liposome internalization analysis within macrophages. dTHP-1 (5 × 10⁵/mL) were stimulated for 18 h with empty liposomes (Lipo), drug-loaded liposomes (LipoRIF) or with polymer-decorated RIF-loaded liposomes (LipoRIF+ Chit LipoRIF+ ε-PLL), all containing calcein. Cells were collected and liposome uptake was analyzed by flow cytometry. Results of Mean Fluorescence Intensity (MFI) are shown as mean ± SD values obtained from two independent experiments.

Figure 6

Polymer-decorated liposomes maintain their antimicrobial effect. To evaluate intracellular bacterial growth, dTHP-1 were infected with Mabs for 3 h at 37 °C at a MOI of 10. After infection, extracellular bacilli were eliminated by 1 h incubation with 250 ug/mL amikacin. Subsequently, dTHP-1 cells were treated for 18 h with unloaded liposomes, free RIF, RIF-loaded liposomes, RIF-loaded Ɛ-PLL-decorated liposomes. Finally, macrophages were lysed with deoxycholate 1% and then samples were diluted in PBS-tween 80 and CFU quantified by plating bacilli in triplicate on 7H10. Replication index was calculated as the ratio between the CFU obtained 18 h of treatment and those obtained immediately after infection, before the addition of the stimuli. The results are shown as mean ± standard deviation of the values obtained from triplicate of each condition and are representative of three different independent experiments. n.s. = non-significant, *** p < 0.001 and **** p< 0.0001 by two-tailed Student’s t-test.