Polymersomes Eradicating Intracellular Bacteria

Abstract

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.

Keywords: drug delivery; intracellular pathogens; polymersomes; tuberculosis; zebrafish.

Figure 1. PMPC polymersomes interaction with phagocytes in vitro.

(a) Real-time imaging of polymersomes entering monocyte-derived macrophages (THP-1 cells) using confocal laser scanning microscopy (CLSM). Note the polymersomes (red signal) are labelled using Cy5, and the macrophage membrane (green signal) is stained using CellMaskTM. (b) Polymersomes uptake measured in 4 different regions of interest (ROI) in (a) plotted as a function of time. (c) Confocal 3D scan of THP-1 cells incubated with Cy5 polymersomes. (d) Immunofluorescence analyses showing no co-localization between polymersomes (red) and EEA1 (green). (e) Live cell imaging of polymersomes (red) and LysoTracker-stained (green) cells. (f) Polymersomes uptake after inhibition of different cellular components: CytochalasinB (actin inhibitor), Dynasore (dynamin inhibitor), Fucoidan (Scavenger Receptors A and B inhibitor), and Polyinosinic acid (Scavenger Receptor A inhibitor and Toll-like 3 receptor agonist ligand stimulator). (t-test comparison with *p < 0.05).

Figure 2. Polymersomes biocompatibility.

(a) Viability assays (MTT) of THP-1 cells incubated with un-loaded and with antibiotic-loaded (rifampicin, isoniazid, and combination of both) polymersomes. Ctrl-: Cells treated with PBS; Ctrl+; DMSO 5%; [polymersomes]: 1 mg/mL; [RIF]: 30 μg/mL; [isoniazid]: 3 μg/mL. (b) Quantitative PCR (qPCR) for analyzing the expression levels of genes involved in cell proliferation (p21 and p53), cell stress (CYP1A1 and CYP1B1), Unfolded Protein Response (ATF4 and ATF6), and oxidative stress (CAT and SOD). (c-e) Representative immunofluorescence imaging to assess Nf-κb-based inflammation in untreated- (c), polymersomes-treated (d), and LPS-treated (e) macrophages. Red: Nf-κb, blue: nucleus. (f) Co-localization quantification of the images in (c-e) using the Pearson's correlation coefficient.

Figure 3. Polymersomes accumulation in macrophages and granulomas in zebrafish embryos infected with M. marinum.

(a). A zebrafish embryo fluorescently labelled with macrophages (red) injected intravenously with M. marinum and the following day with polymersomes containing Cy5 (white). The image was taken 8 hours after polymersomes injection. H, head region; Y, Yolk sac. (b) Quantification of polymersomes uptake over-time in macrophages in zebrafish larvae. (c) enlarged area in (a) where polymersomes are detected within infected macrophages (blue arrows). DLAV, Dorsal longitudinal anastomotic vessel. ISV, intersegmental vessel; CA, Caudal Artery; CV, Caudal Vein. (d) enlarged area in (c) where macrophages containing M. marinum and polymersomes are evident (blue arrows). (e) Zebrafish embryos fluorescently labelled with endothelial cells (green) were injected with M. marinum (red) in the neural tube. Four days later, polymersomes containing Cy5 (white) were injected intravenously and the whole zebrafish was imaged eight hours later. H, Head region; Y, Yolk sac. (f) shows the image in (e) without the signal of green endothelial cells and red M. marinum in order to better observe the selective accumulation of polymersomes (blue arrows) in the granuloma region. The yellow box in (e) is seen enlarged in (g) for observing details of polymersomes accumulation in the granuloma. DLAV, Dorsal Longitudinal Anastomotic Vessel; DA, Dorsal Aorta; PCV, Posterior Cardinal Vein. A graph showing accumulation over time of polymersomes in neural tube granulomas is shown in (h). Scale bars: (a), 300 µm; (c) 100 µm, (d) 25 µm. (e), 300 µm, (f), 300 µm, (g), 50 µm.

Figure 4. Polymersomes eradicating intracellular pathogens in human macrophages.

(a) Average number of drug per single polymersome measured by HPLC (b) THP-1 macrophages infected with S. aureus (M.O.I of 5:1) for 6 hours. Following infection, gentamicin was added to the media to kill extra-cellular bacteria. Macrophages were subsequently treated with polymersomes encapsulating gentamicin, rifampicin, vancomycin, or lysostaphin (all at 1μg/mL). At 6, 22, and 46 hours macrophages were lysed and plated on a BHI agar plate for bacterial colonies to be counted (One-way ANOVA **p < 0.01, ***p < 0.001, error bars = SEM, n = 3). Viability (CFU) analyses of BCG (c), and (d) M. tuberculosis after 24 and 72 hours of incubation with the different formulations (One-way ANOVA with *p < 0.05and **p < 0.01).

Figure 5. Enhanced efficacy of antimicrobials in vivo upon encapsulation in polymersomes.

(a) Zebrafish embryos 2 days post infection were injected with S. aureus (data time 0) followed by a second injection 20 hours later with either PBS, empty polymersomes, free drug and polymersomes loaded with lysostaphin, vancomycin, gentamicin, and rifampicin. Zebrafish were then left for 20 hours before being homogenized and plated on BHI agar for viable colony counts. Graphs show the total number of CFU after treatment (Kruskal-Wallis test with Dunn’s multiple comparison *p <0.05, **p <0.01 and ***p <0.001). (b) Quantification of mCherry expressing M. marinum bacterial burden in zebrafish embryos treated with empty polymersomes, free drugs, and polymersomes loaded with rifampicin, isoniazid, and their combination. (ANOVA test comparison with *p < 0.05).