Cholesterol-Rich Antibiotic-Loaded Liposomes as Efficient Antimicrobial Therapeutics

Abstract

Introduction: Liposomal antibiotics have demonstrated higher bacteriostatic and bactericidal activities than free drugs. In this study, we investigated the effects of cholesterol (Chol) content of liposomes, liposome concentration, and surface coating with polyethylene glycol (PEG) on the antimicrobial activity of moxifloxacin (MOX) liposomes against Staphylococcus epidermidis (ATCC 35984) (S.e).

Methods: MOX-liposome compositions with increasing Chol content were evaluated for their susceptibility to planktonic S.e (growth inhibition, killing, and live-dead staining), as well as against pre-formed biofilms (crystal violet, MTT assay, and confocal microscopy). The MOX-liposomes prepared by active loading were characterized in terms of loading, size distribution, and zeta potential.

Results-discussion: All liposomes had nano-dimensions ranging in diameter from 92nm to 114nm, with zeta-potential values from -2.30mV to -4.50mV. Planktonic bacteria and established biofilms are significantly more susceptible to MOX-liposomes with higher Chol-content than other liposome-types, and the same MOX dose encapsulated in 10 times higher lipids demonstrated higher antimicrobial activity. Coating the MOX liposomes with PEG did not affect their activity. Flow cytometry showed higher binding of Chol-rich liposomes to bacteria, explaining the higher antimicrobial activity. Interestingly, the integrity of calcein-loaded Chol-rich liposomes was much lower than that of liposomes with low or no Chol during incubation with various strains of S. epidermidis. In vivo results in a zebrafish infection model (bacteremia) confirmed the superior activity of Chol-rich MOX-liposomes compared to the free drug.

Conclusion: The current in vitro and in vivo findings demonstrated the potential of PEGylated and Chol-rich liposomal antibiotics as highly efficient therapeutics for the treatment of S. epidermidis infections.

Keywords: Staphylococcus epidermidis; antibiotic; antimicrobial; cholesterol; in vivo; liposomes; moxifloxacin.

Graphical abstract

Figure 1

(A) Growth curves of ATCC 35984 S.e in absence and presence of 0.15µM MOX as free or liposomal drug (MLIP) with varying Chol-contents; empty liposomes (LIP) and mixtures (mix) of empty liposomes (9.5µM lipid) with free MOX were used as control groups. (B) Bacteria growth inhibition (%) by free MOX or MLIPs after 24h incubation. Individual differences are marked by ** for p≤ 0.01, *** for p≤ 0.001, and **** for p ≤ 0.0001.

Figure 2

(A) Time–kill curves of ATCC 35984 S.e, in absence and presence of MOX (0.15µM) as free or liposomal drug (MLIP), with varying Chol-contents. The corresponding total lipid concentration (in each well) is 9.5µM. (B) Effect of MLIP Chol-content on bacteria log Reduction after 24h incubation. (C) Representative images of single plate-serial dilution spotting with S.e (ATCC 35984) corresponding to 10¹–10⁶ dilutions after 24h incubation with free-MOX or MLIP from the time-killing studies for each case. Individual differences are marked by ** for p≤ 0.01, and **** for p ≤ 0.0001.

Figure 3

(A) Flow cytometry images obtained after 24h incubation of ATCC 35984 S.e planktonic bacteria with MOX (0.15µM) as free-MOX or MLIPs (with varying Chol-content); stained live cells (green) and dead cells (red). Total lipid concentration (in each well) is 9.5µM. (B) Live bacterial cells (%) and (C) Dead bacterial cells (%). Asterisks on top of columns denote significant differences from control (plain bacteria) and individual differences are also marked as asterisks. In both cases * is for p≤ 0.05, ** for p≤ 0.01, *** for p≤ 0.001, and **** for p ≤ 0.0001.

Figure 4

Antibiofilm activity on pre-establish biofilms: (A) Biofilm mass and (B) biofilm cell viability of ATCC 35984 S.e bacteria in absence and presence of 0.30µM MOX as free-MOX or MLIPs (liposomes with varying Chol-contents are tested); Empty liposomes (LIP) and Mixtures of empty liposomes (19µM lipid) with free MOX (Mix) are reported as control groups. Individual differences are marked by * for p≤ 0.05), *** for p≤ 0.001, and **** for p ≤ 0.0001.

Figure 5

Antibiofilm activity towards pre-establish biofilms: (A) Confocal microscope images of ATCC 35984 S.e biofilms after 24h incubation with MOX (0.30µM) as free-MOX or MLIPs (with varying Chol-content); stained live cells (green) and dead cells (red). Total lipid concentration (in each well) is 19µM. (B) Dead bacterial cells (%) in total area. Individual differences are marked by ** for p≤ 0.01, *** for p≤ 0.001, and **** for p ≤ 0.0001.

Figure 6

(A) Growth curves, (B) Bacteria growth inhibition (%) after 24h incubation, (C) Time–kill curves, (D) Representative images of plates (of time-kill study), and (E) Bacteria log Reduction values after 24h incubation, of ATCC 35984 S.e in absence and presence of 0.15µM MOX as free-MOX or MLIP-50% with varying lipid concentrations (Low 2.15µM and High 21.5µM); empty liposomes (LIP-50%) and mixtures (MIX) of LIP-50% with free MOX were also used as controls (in A); Individual differences are marked by * symbol when p≤ 0.05, and **** when p ≤ 0.0001.

Figure 7

(A) Flow cytometric dot plots of ATCC 35984 S.e planktonic bacteria after 24h incubation with MOX (0.15µM) as free-MOX or MLIP-50% (with 21.5 µM lipid [High]) with varying PEG contents; Stained live cells (green) and dead cells (red). (B) Dead bacterial cells (%). (C) Growth curves for bacteria in presence and absence of free-MOX or MLIP-50% (High) with varying PEG contents; Symbols **** denote differences where p ≤ 0.0001.

Figure 8

Liposome-Bacteria binding (interaction) (A) FACS histograms representing Rhodamine-positive ATCC 35984 bacterial cells following 24h incubation with LIPs (Rhodamine-lipid incorporating LIPs with varying Chol-contents) at 37°C. Bacterial strains: Se ATCC 35984, Se 11465 (quinolones resistant), Se 762 (quinolones sensitive). Lipid concentration is 0.25mg/mL; bacterial suspension is 4.5x10⁷cfu/mL. (B) Quantitative results. Individual differences are marked by * for p≤ 0.05; and **** for p ≤ 0.0001.

Figure 9

Integrity (%) of Calcein-loaded liposomes during incubation with S.e strains CLIN+ (GRE4388, slime+), CLIN- (GRE2264, slime-), ATCC+ (ATCC 35984, slime+) and ATCC- (ATCC 12228, slime-) at 37°C for a period of 72h. (A) LIP-0; (B) LIP-33%; (C) LIP-50%.

Figure 10

In vivo study in zebrafish. (A) Protocol of studies; (B) Kinetics of infection following zebrafish i.p. injection with Se (reference strain); (C) Bacteremia (cfu/mL) 4h post-treatment with free-MOX and MLIPs. Individual differences are marked by ** for p≤ 0.01, and *** for p ≤ 0.001.