Enhanced penetration and biofilm eradication by sophorolipid micelles encapsulating Honokiol: a comprehensive solution for biofilm-associated lung infections

Abstract

Background: Biofilm-associated lung infections, particularly those caused by Staphylococcus aureus (S. aureus), pose significant clinical challenges to conventional therapies. S. aureus Biofilm infections are refractory to treatment due to the presence of persister bacterial cells and the barrier effect of unique extracellular polymeric substances (EPS).

Results: This study describes the development of multifunctional micelles, HK-SL Ms, utilizing sophorolipid (SL) to encapsulate Honokiol (HK). HK-SL Ms potently disrupted the EPS barrier, killed some internal colonizing bacteria, and inhibited further bacterial adhesion. Consequently, the dynamic cycling of biofilms was hindered, achieving a promising removal of S. aureus biofilms. In vitro studies demonstrated that HK-SL Ms exhibited significant antimicrobial reduction of a 6.42 log₁₀CFU/mL. HK-SL Ms eradicated 71.73% of biofilms by targeting extracellular polysaccharides, extracellular proteins, and viable cells within the biofilm. Additionally, 1.66 log₁₀CFU/mL units of S. aureus within biofilms were killed. Moreover, HK-SL Ms inhibited 91.10% of early S. aureus biofilm formation by obstructing initial bacterial adhesion and the formation of extracellular polysaccharides and polysaccharide intercellular adhesins (PIA). Thus, the reestablishment and reinfection of S. aureus biofilms could be resolved promisingly. Biofilm infections are as predominant in acute pneumonia as in chronic cases, inducing similar lung inflammation. In a murine model of pneumonia infected by S. aureus, HK-SL Ms significantly reduced the bacterial load in the lungs, decreased inflammatory factor levels, and repaired lung tissue damage.

Conclusions: HK-SL Ms offers a novel strategy for the clinical treatment of biofilm-associated infections by dispersing and removing S. aureus biofilms and preventing new infections.

Keywords: Staphylococcus aureus biofilm; Honokiol; Nanomicelle; Sophorolipid.

Fig. 1

Schematic representation of the preparation of HK-SL Ms and their action to Staphylococcus aureus biofilms

Fig. 2

Preparation and characterization of HK-SL Ms. a Schematic diagram of HK-SL Ms preparation. b Particle size distribution of SL Ms. c Particle size distribution of HK-SL Ms. d Potentiograms of HK-SL Ms and SL Ms. e TEM image of HK-SL Ms

Fig. 3

Characterization of HK-SL Ms. a Surface tension curves of SL in water. b Characteristic absorption peaks at 258 nm and 293 nm in the UV spectrum of HK-SL Ms. c X-ray diffraction spectrum of HK-SL Ms. d Hemolysis rate (%) of HK-SL Ms, with 1% Triton X-100 used as a positive control (+) and PBS as a negative control (−)

Fig. 4

Antibacterial activity of HK-SL Ms. a Inhibition rate curve of S. aureus treated with HK-SL Ms for 10 h, where the lowest concentration exhibiting a inhibition rate of 100% is defined as the MIC. Methanol was used as the solvent control group, as it was employed to dissolve HK. b Time-kill curves of S. aureus treated with 16 μg/mL of HK-SL Ms. c Quantitative analysis of S. aureus treated with HK-SL Ms. Different lowercase letters indicate a significant difference (P < 0.05), and the same letters indicate a nonsignificant difference (P > 0.05). d Protein leakage level of S. aureus treated with HK-SL Ms. e SEM images of S. aureus treated with HK-SL Ms. f TEM images of S. aureus treated with HK-SL Ms

Fig. 5

Ability of HK-SL Ms to disperse preformed S. aureus biofilms. a Quantitative results of biofilm dispersion by HK-SL Ms, determined using the crystal violet staining method. Different lowercase letters indicate a significant difference (P < 0.05), and the same letters indicate a nonsignificant difference (P > 0.05). b Quantification of protein dispersion in preformed biofilms by HK-SL Ms. c Quantification of polysaccharide dispersion in preformed biofilms by HK-SL Ms. d Viable bacterial count in preformed biofilms treated with HK-SL Ms

Fig. 6

Confocal images of S. aureus in preformed biofilms treated with HK-SL Ms. All cells were labeled green by SYTO-9, and dead cells were labeled red by PI

Fig. 7

Ability of HK-SL Ms to inhibit early S. aureus biofilm formation. a Inhibition rate of biofilm formation by HK-SL Ms treatment, determined using the crystal violet staining method. Different lowercase letters indicate a significant difference (P < 0.05), and the same letters indicate a nonsignificant difference (P > 0.05). b Inhibition rate of extracellular polysaccharides formation by HK-SL Ms treatment. c Inhibition rate of PIA formation by HK-SL Ms treatment. d Effect of HK-SL Ms on bacterial adhesion, as determined by SEM

Fig. 8

Therapeutic effects on lung infections in mice. a Lung infection modeling process in mice. b HE staining results of lung tissue treated with HK-SL Ms. c–e Anti-inflammatory effects of HK-SL Ms on IL-1β, IL-6, and TNF-α in pulmonary infections. Different lowercase letters indicate a significant difference (P < 0.05), and the same letters indicate a nonsignificant difference (P > 0.05). f Effect of HK-SL Ms on the bacterial load in the lungs