A non-invasive osteopontin-targeted phase changeable fluorescent nanoprobe for molecular imaging of myocardial fibrosis

Fig. 1. (A) Synthetic procedures for OPN-targeted nanoparticles (OPN@PFP-DiR NPs) which loaded perfluoropentane (PFP) for ultrasound (US) and 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) for near-infrared fluorescence (NIR) of molecular imaging, via two step emulsion method and carbodiimide method. (B) Enhanced dual-modal imaging performed by OPN@PFP-DiR NPs via intravenous injection.

Fig. 2. Characteristics of the OPN@PFP-DiR NPs. (A) Macroscopic views of free DiR and OPN@PFP-DiR NPs at the same PLGA concentration in PBS representative SEM images (B) and TEM images (C) of OPN@PFP-DiR NPs. (D) The size distribution of the different PLGA NPs. (E) Size changes of OPN@PFP-DiR NPs at 4 °C after long-term storage. (F) Zeta potential of different PLGA NPs (G) Fluorescence images showed preferential connection between FITC-labeled OPN peptide and DiR-labeled PFP-DiR NPs.

Fig. 3. (A) Optical microscopic image of OPN@PFP-DiR NPs phase transformation by heating from 25 to 50 °C. (B) Optical microscope graphics of phase-transformation after LIFU-irradiated treatment of OPN@PFP-DiR NPs at different frequency (1 W cm−2, 2 W cm−2, 3 W cm−2 and 4 W cm−2) for 3 min. (C and D) NIR images and intensities of OPN@PFP-DiR NPs with different concentrations of DiR. (E and G) US molecular imaging of OPN@PFP-DiR NPs after irradiation by LIFU for 3 min under different condition (1 W cm−2, 2 W cm−2, 3 W cm−2 and 4 W cm−2). (F and H) Gray-scale values of CEUS-mode imaging of OPN@PFP-DiR NPs after LIFU irradiation under different frequency and duration time, as shown in the corresponding figure.

Fig. 4. MI model mice were successfully constructed. (A) ECG before (left) and after (right) modeling. (B) Echocardiograms before (left) and after (right) modeling. Cardiac function indicators including left ventricular internal dimension in diastole (LVIDd) (C), ejection fraction (EF%) (D) and fractional shortening (FS%) (E) were determined by echocardiography.

Fig. 5. The staining outcomes of myocardial fibrosis in myocardial infarction and normal group, myocardial tissues were assessed using various techniques, including (A) HE staining (Aa), Masson staining (Ab), and Sirius red staining (Ac). Notably, the infarcted myocardium exhibited significant collagen fibrosis. (B) FITC-OPN staining was employed to compare myocardial tissue between the myocardial infarction and normal groups. (C) A semiquantitative analysis was conducted to evaluate the staining results of Masson staining, PSR staining, and FITC-OPN staining, comparing the control group with the experimental group.

Fig. 6. The performance of in vivo NIR and US imaging is evaluated. (A) NIR images are obtained. Representative ex vivo fluorescent imaging of mouse organs (heart, lung, liver, kidney, and spleen) at 24 h post-intravenous injections of OPN@PFP-DiR NPs and PFP-DiR NPs. (B) Quantitative analysis of fluorescent intensities (n = 3 per group). The utilization of B-Mode and CEUS imaging (C), as well as (D and E) the assessment of echo intensities before and after LIFU irradiation (3 W cm−2, 10 min) for both PFP-DiR NPs and OPN@PFP-DiR NPs.

Fig. 7. The in vitro and in vivo toxicity and safety assessment of nanoparticles (OPN@PFP-DiR NPs). (A) Morphology of normal H9C2 cells and cardiac fibroblasts (CFs) after co-incubation with OPN@PFP-DiR NPs. Scale bars, 200 μm. (B) The cytotoxicity of NPs on cells was evaluated after a 24 hours incubation period. (C) The main organs, namely the heart, lung, liver, kidney, and spleen, were subjected to HE staining under a light microscope 48 h following the injection of OPN@PFP-DiR NPs. No observable damage was detected in any of the organs.