Relief of Biofilm Hypoxia Using an Oxygen Nanocarrier: A New Paradigm for Enhanced Antibiotic Therapy

Abstract Biofilms are chief culprits of most intractable infections and pose great threats to human health. Conventional antibiotic therapies are hypodynamic to biofilms due to their strong drug resistance, closely related with biofilm hypoxia. A new strategy for enhanced antibiotic therapy by relieving biofilm hypoxia is reported here. A two‐step sequential delivery strategy is fabricated using perfluorohexane (PFH)‐loaded liposomes (lip) as oxygen (O2) carriers (denoted as lip@PFH@O2) and commercial antibiotics. The results indicate that the two‐step sequential treatment exhibits much lower minimum bactericidal concentrations than the antibiotic treatment alone. In this design, the lip@PFH@O2 holds positively charged surface for better biofilm penetration. After penetrating into biofilm, oxygen can be released from lip@PFH@O2 by inches, which greatly relieves biofilm hypoxia. With the relief of hypoxia, the quorum sensing and the drug efflux pumps of bacteria are suppressed by restraining related gene expression, leading to the reduced antibiotic resistance. Furthermore, the in vivo experimental results also demonstrate that lip@PFH@O2 can effectively relieve biofilm hypoxia and enhance therapeutic efficacy of antibiotics. As a proof‐of‐concept, this research provides an innovative strategy for enhanced antibiotic therapy by relieving hypoxia, which may hold a bright future in combating biofilm‐associated infections.


Measurement of PFH loading amount in lip@PFH@O 2 :
The PFH loading capacity in lip@PFH@O 2 is evaluated by gas chromatography-mass spectrometry (GC-MS) referring to the literature protocol. [1] lip@PFH nanoparticles (10, 20, 30, 40 μL) are dissolved in acetonitrile (40 μL) and vortex-mixed for 2 minutes. Then, 1,1,1,3,3-pentafluorobutane (200 μL) is added into each sample and the mixture is vortex-mixed for 5 minutes. The mixture is centrifuged (3000 rpm) for 2 min at 4 ℃ and then frozen at -30 ℃ for 2 hours. The lower phase is put into another 2 mL tube for GC-MS measurement and the upper phase is withdrawn.
Culturing and harvesting P. aeruginosa biofilm: Pseudomonas aeruginosa (abbreviated to P. aeruginosa, ATCC 27853, purchased from Guangdong culture collection center) is used in this research. At first, P. aeruginosa suspension (10 8 cfu/mL, 100 μL) and LB broth medium (100 μL) are placed into 24 well plates. The 24 well plates are then put into the nitrogen bag.
Subsequently, the 24 well plates in nitrogen bag are put into the incubator to culture at 37 °C.
After 24 hours, the timeworn medium is replaced by fresh LB broth medium and the biofilm 3 is grown in nitrogen condition for another 24 hours. Finally, the medium is removed and the P. aeruginosa biofilm attached on 24 well plates is obtained.
Penetration of lip@PFH into P. aeruginosa biofilm: Laser scanning confocal microscope (OLYMPUS IX83-FV3000) is employed to evaluate the penentration ability of nanoparticles into biofilms. Firstly, the lip@PFH is fluorescent marked by encapsulating the chlorin e6 (Ce6) inside. Next, the lip@PFH@Ce6 with red fluorescence under 561 nm exciting light is put into the P. aeruginosa biofilm for one hour. Afterwards, the biofilm is washed for three times with PBS solution to wash off the nanocarriers on the surface of biofilm and then the biofilm is stained by the SYTO 9, which can dye the live bacteria green, for 15 min in the dark.Then, the superfluous dye is wash off by PBS solution. At last, the biofilm is observed under laser scanning confocal microscope.
In vivo immunofluorescence staining of biofilm infected subcutaneous abscess by hypoxyprobe: At first, different nanoparticles (100 μL) are added into the P. aeruginosa biofilms infected subcutaneous abscess for two hours respectively. Then, the pimonidazole hydrochloride (100 μL) is injected into the subcutaneous abscess for 90 minutes incubation.
After incubation, the mice are sacrificed and the frozen abscess slices are stained through a reported procedure. [2] Finally, the slices are observed under a fluorescence microscope (Olympus IX81).
The in vivo qRT-PCR gene detection assay of bacteria in subcutaneous abscess: Firstly, different nanoparticles (100 μL) are injected into the P. aeruginosa biofilm infected subcutaneous abscess for two hours respectively. Then, the mice are sacrificed and the bacteria in abscess is obtained by centrifugation (3000 rpm, 5 min). Subsequently, the obtained bacteria is detected by the qRT-PCR. Before detection, the bacterie amount in each sample is quantified by the standard plate counting assay to guarantee the bacteria amount is same in each group.
Statistical Analysis: Data were expressed as mean ± SD and the statistical significance is determined using one-way ANOVA analysis. *P < 0.05, **P < 0.01.