Targeted delivery of TAPI-1 via biomimetic nanoparticles ameliorates post-infarct left ventricle function and remodelling

Figures

• 

  Figure 1. Fabrication and characterization of Neu-LNPs. (A) Schematic outlining the procedure of Neu-LNPs preparation. The characteristics of Neu-LNPs with varying ratios of neutrophil membrane proteins to LNPs were assessed by size (B), zeta potential (C), and PDI (polymer dispersity index, D). *P < 0.05 vs. Neu-Protein, †P < 0.05 vs. Neu-LNPs (0.5:1). (E) Transmission electron microscopy (TEM) images of typical LNPs and Neu-LNPs. Scale bars: 50 nm. (F) Grouped data showing TEM analysis of the density size of LNPs and Neu-LNPs at 0, 7, and 14 days indicate size stabilization within a two-week duration despite a difference in diameter between LNPs and Neu-LNPs (*P < 0.05). (G) Fourier-transform infrared (FTIR) spectroscopy analysis of neutrophil membrane, LNPs and Neu-LNPs. (H) Cytotoxicity of Neu-LNPs assessed by cell viability in HUVECs and H9C2 cells. Data set was analysed with one-way ANOVA followed by Tukey’s multiple comparisons for B–D and H and with unpaired Student’s t-test for F. n = 5–6 in each group.

• 

  Figure 2. Assessment of cellular uptake for Neu-LNPs. (A) Three-dimensional confocal images illustrating the cellular internalization of Neu-LNPs with varying ratios of neutrophil membrane proteins to LNPs in HUVECs. Flow cytometric evaluation of cellular uptake of Neu-LNPs in HUVECs, presented as flow cytometry images (B) and grouped data shown as fluorescence intensity (C). (D) Three-dimensional confocal images illustrating the cellular internalization of Neu-LNPs with varying ratios of neutrophil membrane proteins to LNPs in H9C2s. Flow cytometric evaluation of cellular uptake of Neu-LNPs in H9C2s, presented as flow cytometry images (E) and grouped data (F). Scale bars: 20 µm. n = 5 in each group, *P < 0.05 vs. black, †P < 0.05 vs. Neu-Protein: LNPs 0.1:1. Data set was analysed with one-way ANOVA followed by Tukey’s multiple comparisons for C and F.

• 

  Figure 3. Targeting and binding analysis of Neu-LNPs in vitro. (A) Schematic representation of the endothelial-inflamed cardiomyocytes Transwell model utilized for targeting and binding analysis of Neu-LNPs. (B, C) Quantitative data depicting the cellular uptake of LNPs/Neu-LNPs by apical HUVECs or basolateral H9C2s using flow cytometric analysis, presented as fluorescence mean. (D) Western blot analysis confirming the presence of CXCR4 and CD11a in Neu-LNPs. (E, F) Confocal images showing the up-regulated expression of ICAM-1 and enhanced uptake of Neu-LNPs by LPS-stimulated HUVECs or H9C2s, with inhibition of LFA1/Mac-1 by anti-CD18 antibody leading to reduced uptake. ICAM-1, intercellular cell adhesion molecule-1; LFA1, lymphocyte function-associated antigen 1; Mac-1, macrophage-1 antigen. Scale bars: 10 µm. n = 5 in each group, *P < 0.05 vs. control, †P < 0.05 vs. LNPs + LPS or LNPs. Data set was analysed with one-way ANOVA followed by Tukey’s multiple comparisons for B and C.

• 

  Figure 4. Targeting and cellular uptake analysis of Neu-LNPs in vivo. (A) Confocal images showing the distribution of Dil-labelled Neu-LNPs in the LV myocardium of SHAM and MI rats administered with Neu-LNPs or MI rats administered with Neu-LNPs incubated with anti-CD18 antibody. Scale bars: 50 µm. n = 3 in each group. (B, C) Confocal immunostaining images displaying the colocalization of Dil-labelled Neu-LNPs with macrophages (B) and cardiomyocytes (C) in the peri-infarct regions of MI rat hearts. Nuclei were stained with DAPI. Scale bars: 25 µm.

• 

  Figure 5. In vivo biodistribution analysis of Neu-LNPs. (A) In vivo small animal imaging exhibits the biodistribution of Dir-labelled LNPs/Neu-LNPs in MI or SHAM rats at different time points within 48 h post-injection. (B) Grouped data illustrating quantified fluorescence intensity of Dir-labelled LNPs/Neu-LNPs in the heart at distinct time intervals within the initial 48 h post-injection. (C, D) Ex vivo fluorescence image and quantification of fluorescence intensity of the heart at 48 h after LNPs/Neu-LNPs injection. n = 3 in each group, *P < 0.05 vs. MI/Dir, †P < 0.05 vs. SHAM/Neu-LNPs, ‡P < 0.05 vs. MI/LNPs. Data set was analysed with two-way ANOVA followed by Sidak’s multiple comparisons for B and with one-way ANOVA followed by Tukey’s multiple comparisons for D.

• 

  Figure 6. Effect of TAPI-1-Neu-LNPs on the LV function and cardiac dynamics. (A) Schematic outlining the experimental design and measurement protocols. (B) TACE activity in LV myocardium in SHAM rats treated with vehicle (VEH) and MI rats treated with VEH, Neu-LNPs, or TAPI-1-Neu-LNPs. (C) TNF-α levels in plasma and LV myocardium in SHAM rats treated with vehicle (VEH) and MI rats treated with VEH, Neu-LNPs, or TAPI-1-Neu-LNPs. (D) Echocardiographic evaluation of LVESV, LVEDV, LVEF, and IZ. (E) Analysis of cardiac haemodynamics on HR, SBP, LVEDP, LVPSP, and maximum rates of pressure change during isovolumetric contraction (+dP/dtmax) and relaxation (−dP/dtmax) across six experimental groups. LVESV, left ventricular end-systolic volume; LVEDV, LV end-diastolic volume; LVEF, left ventricular ejection fraction; IZ, ischaemic zone; HR, heart rate; SBP, systolic blood pressure; LVEDP, left ventricular end-diastolic pressure; LVPSP, left ventricular peak systolic pressure. n = 5–7; #P < 0.05, 4 weeks vs. 24 h; *P < 0.05 vs. SHAM/VEH; †P < 0.05, MI/TAPI-1-Neu-LNPs vs. MI/VEH, MI/Neu-LNPs, and/or MI/TAPI-1. Data set was analysed with one-way ANOVA followed by Tukey’s multiple comparisons for B, C,…

• 

  Figure 7. Effect of the TAPI-1-Neu-LNPs on cardiac remodelling. (A) Representative images of heart and lung specimens from six experimental groups following a 4-week treatment. (B, C) Anatomic assessments of heart weight-to-body weight ratio and lung weight-to-body weight ratio. (D) Representative Masson’s trichrome staining images showing cardiac fibrosis from SHAM or HF rats treated with vehicle (VEH), Neu-LNPs, TAPI-1-LNPs/Neu-LNPs, or TAPI-1 alone. (E) Grouped data showing the quantified collagen deposition (%) of the heart. (F–H) Comparative quantification of mRNA expression of pro-fibrotic markers collagen-I (F), collagen-III (G), and fibronectin (H) in the peri-infarct regions of the left ventricle across six treatment cohorts. Values are mean ± S.E.M. (n = 6–7 for each group). *P < 0.05 vs. SHAM/VEH; †P < 0.05 vs. MI/VEH. Data set was analysed with one-way ANOVA followed by Tukey’s multiple comparisons for B, C, and E–H.