Mucosomes as next-generation drug carriers for treating mucus-resident bacterial infections and biofilms

Abstract

Deaths connected to bacterial infections are expected to outnumber those caused by cancer by 2050. Multiple advantages, including enhanced efficacy of the treatment, characterize the use of nanocarriers to deliver antibiotics. This work explores the use of mucosomes - intrinsically glycosylated mucin nanoparticles - to deliver ciprofloxacin to fight Pseudomonas aeruginosa and Staphylococcus aureus infections. Mucins are a family of glycoproteins representing the major non-aqueous component of human mucus and are known for actively interacting with bacteria, reducing their virulence, and limiting their aggregations. This study shows that these critical properties of mucin are preserved in mucosomes, enabling a strong synergy with the loaded antimicrobial drug. Empty mucosomes exert a bacteriostatic activity, inhibiting bacterial growth up to 70%. Ciprofloxacin-loaded mucosomes were able to decrease the minimum inhibitory concentration of ciprofloxacin against S. aureus by up to 50%. Mucosomes could prevent biofilm formation and disassemble well-established biofilms by reducing the biomass by up to 98%. Mucosomes further facilitated the transmucosal delivery of ciprofloxacin in a 3D mucus-mimicking model. These results, together with the possibility of freeze-drying and storing drug-loaded mucosomes without impairing their efficacy, suggest the suitability of this approach to tackle mucosal bacterial infections. Interestingly, this nanosystem has been shown to enhance the phagocytic action of blood in eradicating bacterial biofilms.

Keywords: Ciprofloxacin; Drug delivery; Glycans; Infections; Mucin; Mucosa; Nanoparticles.

Fig. 1

Characterization of the ciprofloxacin-loaded mucosomes. (a) Transmission electron microscopy images representing the size and the morphology of both empty and ciprofloxacin-loaded mucosomes; images acquired at 100 k× magnification (scale bar = 200 nm). The red dashed circumference was added subsequently to better highlight the perimeter of mucosomes. (b) Release kinetics of ciprofloxacin from mucosomes into PBS at pH = 7.4. c) Photograph taken 3 h after suspending free ciprofloxacin (1.0 mg/mL) in different solvents (dH₂O with or without 5% or 10% DMSO), showing noticeable Active Pharmaceutical Ingredient sedimentation in the free ciprofloxacin samples, in contrast to the absence of deposition in cipro-loaded NP suspensions.

Fig. 2

Efficacy of ciprofloxacin-loaded mucosomes against planktonic cultures of P. aeruginosa and S. aureus. Results expressed in terms of inhibition radius for the agar diffusion tests performed on (a) P. aeruginosa and (b) S. aureus. Empty mucosomes were found effective in limiting the growth of (c) P. aeruginosa and (d) S. aureus starting from a treatment concentration of 64 µg/mL and 1.0 µg/mL, respectively (these values indicate the concentration of ciprofloxacin that would be delivered by the employed amount of mucosomes if they were loaded with drug). When P. aeruginosa was treated with ciprofloxacin-loaded mucosomes, no significant differences between the efficacy of the mucosomal formulation and the free active principle. (e) At a MIC-equivalent concentration (0.25 µg/mL) and (f) at higher concentrations (512 µg/mL). Conversely, ciprofloxacin-loaded mucosomes were found to be more effective than free ciprofloxacin against S. aureus, both (g) at the MIC-equivalent concentrations (0.50 µg/mL), as well as at h) higher concentrations (512 µg/mL).

Fig. 3

Ciprofloxacin-loaded mucosomes can be freeze-dried and stored before being used. No differences in terms of antibacterial efficacy were found for (a) P. aeruginosa and (b) S. aureus, when comparing the survival rate of the cultures to freeze-dried and rehydrated ciprofloxacin-loaded mucosomes, ciprofloxacin-loaded mucosomes, and free ciprofloxacin. The concentrations of active principle used for this experiment were chosen as the minimum concentration employed in this work, the MIC of ciprofloxacin according to the EUCAST database, and the maximum concentration among those used in this work at which all the treatments showed the same efficacy on both bacterial strains.

Fig. 4

Evaluation of the ability of mucosomes to prevent biofilm formation. (a, e) Dose-dependent quantitative effect of each treatment on P. aeruginosa (a) and S. aureus (e). (b, f) SEM on P. aeruginosa (b) and S. aureus (f) biofilms. Images acquired at 6k× magnification (scale bars = 2 μm), insets at 15k× (scale bar = 1 μm). (c, g) CLSM 3D projections of P. aeruginosa (c) and S. aureus (g) biofilms. The images acquired at 63× magnification (scale bars = 50 μm) highlight the biofilm matrix (stained in cyan), viable bacteria (stained in green), and dead bacteria (stained in red). (d, h) Quantification of the blue-channel fluorescence intensity for P. aeruginosa (d) and S. aureus (h) biofilms from CLSM acquisitions.

Fig. 5

Evaluation of the ability of mucosomes to disrupt established biofilms. (a, e) Dose-dependent quantitative effect of each treatment on P. aeruginosa (a) and S. aureus (e) biofilms. (b, f) SEM on P. aeruginosa (b) and S. aureus (f) biofilms. Images acquired at 6k× magnification (scale bars = 2 μm), insets at 15k× (scale bar = 1 μm). (c, g) CLSM 3D projections of P. aeruginosa (c) and S. aureus (g) biofilms. The images acquired at 63× magnification (scale bars = 50 μm) highlight the biofilm matrix (stained in blue), viable bacteria (stained in green), and dead bacteria (stained in red). (d, h) Quantification of the blue-channel fluorescence intensity for P. aeruginosa (d) and S. aureus (h) biofilms from CLSM acquisitions.

Fig. 6

Mucosomes enhance the delivery of ciprofloxacin through a mucus-like interface. (a) Schematization of the experimental set-up. (b) When ciprofloxacin was loaded into mucosomes, the presence of a mucus-mimicking interface did not alter its diffusion into the acceptor compartment. The survival rate of (c) P. aeruginosa and (d) S. aureus significantly decreases (p < 0.001 for P. aeruginosa and p < 0.00001 for S. aureus) when the cultures are treated with ciprofloxacin-loaded mucosomes, compared to free ciprofloxacin.

Fig. 7

The synergistic effect between mucosomes and human blood in eradicating P. aeruginosa and S. aureus biofilms. Heat map illustration of the logarithmic decrease in (a) P. aeruginosa and (b) S. aureus viability following treatment with blood samples from six human healthy donors, as well as with blood enriched with empty mucosomes (NPs), free ciprofloxacin (Cip), and ciprofloxacin-loaded mucosomes (NPs_Cip). All data for both bacterial strains were statistically significant vs. CTRL (P value < 0.05).

Fig. 8

Mucosomes colocalize with bacteria in a 3D model of pulmonary mucus infection. (a) The red channel of the image shows P. aeruginosa that was transformed to express red fluorescence (mCherry). From this picture, it is possible to observe the bacterial aggregates (highlighted with green circles) that were formed during the experimental time-course. (b) The green channel of the image for P. aeruginosa culture shows a non-random distribution of FITC-loaded mucosomes (clusters are highlighted with red circles). (c) By merging the red and green channels it is possible to appreciate the co-localization between bacteria clusters and mucosomes (highlighted with green circles). (d) Similarly, by looking at the red channel image for S. aureus cultures (bacteria were transformed to express DsRed fluorescent protein), it can be noticed that bacteria are organized into multicellular clusters (highlighted with green circles) after 48 h in culture. (e) From the green channel image, it is possible to spot different aggregates of mucosomes (highlighted with red circles). (f) When merging the 2 channels also for S. aureus cultures, mucosomes are preferentially located in the proximity of S. aureus clusters. All the scale-bars in this figure are equal to 50 μm.