Molecular ultrasound imaging of neutrophil membrane-derived biomimetic microbubbles for quantitative evaluation of hepatic ischemia-reperfusion injury

Rationale: Early diagnosis of hepatic ischemia-reperfusion injury (HIRI), the major cause of early allograft dysfunction or primary non-function, is critical in orthotopic liver transplantation. However, liver biopsy is still the primary method for HIRI evaluation in clinical practice despite its numerous complications and shortcomings such as hemorrhage and inaccuracy. Herein, we aimed to develop a non-invasive, highly accurate, and specific method for detecting HIRI. Methods: We developed a top-down and bottom-up strategy to fabricate neutrophil biomimetic microbubbles (MBneu). Neutrophil membrane was mixed with liposomes at a defined mass ratio by sonication. The air in the vial was exchanged with perfluoropropane, and then the solution was mechanically vibrated to form MBneu. Results: MBneu retained the neutrophil proteins, preferentially targeted inflamed hepatic tissue in a rat model of HIRI, and demonstrated physicochemical properties typical of liposome-based MBs because of its artificial phospholipid content. With MBneu we can quantitively evaluate the severity of HIRI, which is helpful for early diagnosis and the prediction of outcome. In addition, MBneu was shown to be safe and showed no immunogenicity. Conclusion: We demonstrated molecular ultrasound imaging of HIRI with MBneu. This new synthesis strategy may be applied to different clinical scenarios using other cell types in the future.


Characterization of MBs
DLS measurements were collected at 25 ℃ on a 90 Plus/BI-MAS instrument (Brookhaven Instruments Corporation, USA). Three measurements for each sample were averaged.
To confirm the presence of neutrophil membrane in the MBneu shell, the synthetic liposomes were fluorescently labeled by addition of 1μg DiO in the lipid mixture before liposome formation, and 1μg DiI was used to track the neutrophil membrane.
Fluorescence images were acquired on an inverted fluorescence microscope (DMI 4000B, Leica, Germany).
To detect neutrophil membrane proteins in MBneu, western blotting analysis was performed. 500 μL of MBneu, neutrophil membrane (neutrosome), or neutrophil were lysed by addition of 200 μL of RIPA lysis buffer and 2 μL of phenylmethylsulfonyl fluoride (Solarbio, China) and incubated at 4 ℃ for 30 min. The mixture was then centrifuged at 2000 rcf for 2 min and the supernatant was collected. The concentration of protein in the supernatant was determined using a BCA kit (ThermoFisher, USA).

Immunohistochemistry
Liver sections were deparaffinized with xylene and alcohol and then incubated in 10 mM citrate buffer (pH 6.0) for 10 min at 90 ℃. Then, 3% hydrogen peroxide in methanol was added to the sections at 4 ℃ for 30 min to inactivate endogenous peroxidases. The tissues were treated with 10% normal horse serum for antigen Finally, the nuclei were stained with hematoxylin.

Biosafety and immunogenicity of MBneu
Healthy rats were anesthetized with 2% pentobarbital sodium (2 mL/kg i.p.) then randomly administered 500 μL of MBneu or MBcon in PBS at a concentration of 1×10 8 MB/mL (100 times the imaging dosage) or the same volume of PBS via the tail vein (n = 5). Blood samples were collected 1 and 7 days after injection. The rats were sacrificed 7 days after injection and blood and major organs (heart, liver, spleen, lung, kidney) were harvested to evaluate the biosafety of the MBs. Liver (AST, ALT) and kidney (Cr, BUN) function biomarkers in serum were measured using a clinical chemistry analyzer (Hitachi 7150). The concentration of cytokines (IL-1β, IL-6, TNFα, IL-2, IL-4, IFN-γ) was determined by enzyme-linked immunosorbent assay (R&D Systems Inc., USA) according to the manufacturer's procedure. Organs were stained with H&E.

Distribution of MBneu
Rats were anesthetized with 2% pentobarbital sodium (2 mL/kg i.p.) then administered 50 μL of DiR-labelled MBneu or MBcon at a concentration of 1×10 7 MB/mL via the tail vein. The rats were imaged using an in vivo fluorescence imaging system (IVIS, PerkinElmer, USA) in luminescence imaging mode. Then the rats were sacrificed and the major organs (heart, liver, spleen, lung, kidney) were harvested for ex vivo imaging to determine the distribution of the MBs.

In vivo US imaging of MB circulation time
Targeted CEUS was performed using a clinical ultrasound imaging system (EPQ7 digital premium ultrasound system, PHILIPS, Netherlands) with a high-frequency linear array transducer operating with the following parameters: mechanical index, 0.08; frequency, 10 MHz; imaging depth, 3-4 cm. All imaging settings were kept constant throughout the imaging sessions for all animals. Healthy rats were administered 50 μL of MBcon or MBneu in PBS at a concentration of 1×10 7 MB/mL via the tail vein and then the MBs lifespan was recorded (n = 5). of MBcon was 7 min, while that of MBneu was ~6 min; however, there was no statistically significant difference between the two groups. So, the circulation time of the newly developed MBneu was ~6-7 min.