PEG-GVs

Stages

1. 1.
   Baseline contrast-agent comparison in carotid artery(broad_screen)

   This stage compares available contrast formulations to establish whether nanoscale GVs can image the vascular wall and whether PEG modification improves persistence.

   Selection: Ability of GVs, SonoVue, and PEG-GVs to generate carotid artery wall contrast, including signal stability and duration.

2. 2.
   Target-biomarker confirmation in plaques(functional_characterization)

   This stage establishes that CXCR4 is present in plaques and low in normal vessels, supporting the rationale for a CXCR4-targeted probe.

   Selection: Demonstration of CXCR4 expression in atherosclerotic plaques by flow cytometry and immunofluorescence.

3. 3.
   Targeted binding and in vivo imaging evaluation(confirmatory_validation)

   This stage tests whether adding CXCR4 targeting translates from biomarker rationale into measurable cell binding and stronger plaque imaging in animals.

   Selection: Cell adhesion and in vivo ultrasound imaging evidence for targeting performance of CXCR4-GVs.

4. 4.
   Plaque localization and safety assessment(secondary_characterization)

   This stage checks whether the targeted vesicles physically localize within vulnerable plaques and whether the formulations appear safe by the reported assays.

   Selection: Fluorescent plaque scanning for localization plus CCK8, H&E, and serum testing for safety.

Steps

1. 1.
   Compare GVs, SonoVue, and PEG-GVs in carotid artery imagingcontrast agents under comparison

   Establish baseline vascular-wall imaging capability and persistence differences among contrast formulations.

   The study first needs to show that nanoscale GVs can image the vascular wall and whether PEG modification improves signal duration before evaluating targeted plaque imaging.

2. 2.
   Measure CXCR4 expression in plaques by flow cytometry and immunofluorescence

   Confirm that CXCR4 is enriched in atherosclerotic plaques relative to normal vessels.

   Target-expression confirmation provides the biological rationale for constructing and testing a CXCR4-directed probe.

3. 3.
   Test CXCR4-GV binding to ox-LDL-induced RAW264.7 cellstargeted probe being evaluated

   Assess whether the targeted vesicles bind a plaque-relevant macrophage cell model.

   A cell-based binding assay is a lower-complexity test of targeting behavior before or alongside in vivo plaque imaging.

4. 4.
   Compare plaque imaging signal of CXCR4-GVs versus Con-GVs in animalstargeted probe being benchmarked in vivo

   Determine whether CXCR4 targeting improves plaque imaging signal strength and durability in animals.

   After establishing target rationale and cell binding, in vivo imaging tests whether those properties translate into improved plaque visualization.

5. 5.
   Scan plaques after fluorescent vesicle injection to assess localization

   Visualize whether vesicles pass through plaque neovasculars and accumulate in vulnerable plaques.

   Localization imaging provides mechanistic support for the in vivo ultrasound signal by showing physical access and accumulation within plaques.

6. 6.
   Assess safety with CCK8, H&E staining, and serum detectionformulations undergoing safety evaluation

   Evaluate whether GVs, PEG-GVs, and CXCR4-GVs show acceptable safety in the reported assays.

   Safety testing is needed after imaging-performance evaluation to determine whether the candidate probe remains suitable for further use.